Bacteria are the oldest, structurally simplest, and most abundant forms of life on earth. Its detection has always been a serious question since the emerging of modern science and technology. There has been a phenomenal growth in the field of real-time bacteria detection in recent years with emerging applications in a wide range of disciplines, including medical analysis, food, environment and many more. Two important analytical functions involved in bacteria detection are cell lysis and polymerase chain reaction (PCR). Cell lysis is required to break cells open to release DNA for use in PCR. PCR is required to reproduce millions of copies of the target genes to reach detection limit from a low DNA concentration. Conventionally, cell lysis and PCR are performed separately using specialized equipments. Those bulky machines consume much more than needed chemical reagents and are very time consuming. An efficient, cost-effective and portable solution involving Nanotechnology and Lab-on-a-Chip (LOC) technology was proposed. The idea was to utilize the excellent antibacterial property of surface-functionalized nanoparticles to perform cell lysis and then to perform PCR on the same LOC system without having to remove them from the solution for rapid detection of bacteria. Nanoparticles possess outstanding properties that are not seen in their bulk form due to their extremely small size. They were introduced to provide two novel methods for LOC cell lysis to overcome problems of current LOC cell lysis methods such as low efficiency, high cost and complicated fabrication process. The first method involved using poly(quaternary ammonium) functionalized gold and titanium dioxide nanoparticles which were demonstrated to be able to lyse E. coli completely in 10 minutes. The idea originated from the excellent antibacterial property of quaternary ammonium salts that people have been using for a long time. The second method involved using titanium dioxide nanoparticles and a miniaturized UV LED array. Titanium dioxide bears photocatalytic effect which generates highly reactive radicals to compromise cell membranes upon absorbing UV light in an aqueous environment. A considerable reduction of live E. coli was observed in 60 minutes. The thesis then evaluates the effect of nanoparticles on PCR to understand the roles nanoparticles play in PCR. It was found that gold and titanium dioxide nanoparticles induce PCR inhibition. How size of gold nanoparticles affected PCR was studied as well. Effective methods were discovered to suppress PCR inhibition caused by gold and titanium dioxide nanoparticles. The pioneering work paves a way for the integration of nanoparticle cell lysis and LOC PCR for rapid detection of bacteria. In the end, an integrated system involving nanoparticle cell lysis and microchip PCR was demonstrated. The prototyped system consisted of a physical microchip for both cell lysis and PCR, a temperature control system and necessary interface connections between the physical device and the temperature control system. The research explored solutions to improve PCR specificity in a microchip environment with gold nanoparticles in PCR. The system was capable of providing the same performance while reducing PCR cycling time by up to 50%. It was inexpensive and easy to be constructed without any complicated clean room fabrication processes. It can find enormous applications in water, food, environment and many more.

Bacteria are the oldest, structurally simplest, and most abundant forms of life on earth. Its detection has always been a serious question since the emerging of modern science and technology. There has been a phenomenal growth in the field of real-time bacteria detection in recent years with emerging applications in a wide range of disciplines, including medical analysis, food, environment and many more. Two important analytical functions involved in bacteria detection are cell lysis and polymerase chain reaction (PCR). Cell lysis is required to break cells open to release DNA for use in PCR. PCR is required to reproduce millions of copies of the target genes to reach detection limit from a low DNA concentration. Conventionally, cell lysis and PCR are performed separately using specialized equipments. Those bulky machines consume much more than needed chemical reagents and are very time consuming. An efficient, cost-effective and portable solution involving Nanotechnology and Lab-on-a-Chip (LOC) technology was proposed. The idea was to utilize the excellent antibacterial property of surface-functionalized nanoparticles to perform cell lysis and then to perform PCR on the same LOC system without having to remove them from the solution for rapid detection of bacteria. Nanoparticles possess outstanding properties that are not seen in their bulk form due to their extremely small size. They were introduced to provide two novel methods for LOC cell lysis to overcome problems of current LOC cell lysis methods such as low efficiency, high cost and complicated fabrication process. The first method involved using poly(quaternary ammonium) functionalized gold and titanium dioxide nanoparticles which were demonstrated to be able to lyse E. coli completely in 10 minutes. The idea originated from the excellent antibacterial property of quaternary ammonium salts that people have been using for a long time. The second method involved using titanium dioxide nanoparticles and a miniaturized UV LED array. Titanium dioxide bears photocatalytic effect which generates highly reactive radicals to compromise cell membranes upon absorbing UV light in an aqueous environment. A considerable reduction of live E. coli was observed in 60 minutes. The thesis then evaluates the effect of nanoparticles on PCR to understand the roles nanoparticles play in PCR. It was found that gold and titanium dioxide nanoparticles induce PCR inhibition. How size of gold nanoparticles affected PCR was studied as well. Effective methods were discovered to suppress PCR inhibition caused by gold and titanium dioxide nanoparticles. The pioneering work paves a way for the integration of nanoparticle cell lysis and LOC PCR for rapid detection of bacteria. In the end, an integrated system involving nanoparticle cell lysis and microchip PCR was demonstrated. The prototyped system consisted of a physical microchip for both cell lysis and PCR, a temperature control system and necessary interface connections between the physical device and the temperature control system. The research explored solutions to improve PCR specificity in a microchip environment with gold nanoparticles in PCR. The system was capable of providing the same performance while reducing PCR cycling time by up to 50%. It was inexpensive and easy to be constructed without any complicated clean room fabrication processes. It can find enormous applications in water, food, environment and many more.

Archaea is one of the three domains of life and studies of archaeal biology are important for understanding of life in extreme environments, fundamental biogeochemical processes, the origin of life, the eukaryotic cell and their own, unique biology. This thesis presents four studies of the archaeal cell, using the extremophilic Sulfolobus and ocean living Nitrosopumilus as model systems. Cell division in crenarchaea is shown to be carried out by a previously unknown system named Cdv (cell division). The system shares homology with the eukaryotic ESCRT-III system which is used for membrane reorganization during vesicle formation, viral release and cytokinesis. Organisms of the phylum Thaumarchaeota also use the Cdv system, despite also carrying genes for the euryarchaeal and bacterial cell division system FtsZ. The thaumarchaeal cell cycle is demonstrated to be dominated by the prereplicative and replicative stage, in contrasts to the crenarchaeal cell cycle where the cell at the majority of the time resides in the postreplicative stage. The replication rate is remarkably low and closer to what is measured for eukaryotes than other archaea. The gene organization of Sulfolobus is significantly associated with the three origins of replication. The surrounding regions are dense with genes of high importance for the organisms such as highly transcribed genes, genes with known function in fundamental cellular processes and conserved archaeal genes. The overall gene density is elevated and transposons are underrepresented. The archaeal virus SIRV2 displays a lytic life style where the host cell at the final stage of infection is disrupted for release of new virus particles. The remarkable pyramid-like structure VAP (virus associated pyramids), that is formed independently of the virus particle, is used for cell lysis. The research presented in this thesis describes unique features of the archaeal cell and influences our understanding of the entire tree of life.

Le travail de thèse présenté dans ce manuscrit s’inscrit dans une dynamique d’intégration de matériaux non conventionnels en systèmes microfluidiques. Il vise à démontrer le potentiel du cPDMS, un matériau nanocomposite formé d’une matrice de polydiméthylsiloxane rendu conducteur par l’ajout de nanoparticules de carbone. Compatible avec les procédés technologiques habituels, le cPDMS peut être structuré dans une large gamme d’épaisseurs et de géométries mais présente surtout l’avantage de pouvoir être collé irréversiblement sur verre, PDMS et silicium. Son intégration est parfaitement étanche, rapide à mettre en oeuvre, et très économique. La première partie du manuscrit est consacrée à la caractérisation de ce matériau. Ses propriétés électriques et de surface, pouvant être critiques pour une utilisation en microfluidique, ont été particulièrement étudiées. Les champs électriques offrant de nombreuses possibilités pour réaliser des fonctions clés en microfluidique (détection, séparation, manipulation de fluides ou de particules), nous avons choisi d’évaluer l’intérêt d’électrodes de cPDMS dans deux types d’applications. Les aspects de détection ont d’abord été mis en évidence à l’aide de mesures électrochimiques. Cette méthode a permis à la fois de caractériser la surface du cPDMS tout en validant son utilisation potentielle pour des applications d’analyses électrochimiques. Dans la dernière partie du manuscrit, le matériau a été testé pour la manipulation de particules à travers l’observation de différents phénomènes électrocinétiques. Ceux-ci ont conduit à la mise au point de dispositifs microfluidiques (intégrant des lectrodes de cPDMS) dédiés à la lyse et à l’électrofusion de cellules. / The work presented in this thesis deals with the integration of non-conventional materials in microfluidic systems. It aims to demonstrate the potential of cPDMS, a conductive nanocomposite material made up of polydimethylsiloxane matrix mixed with carbon nanoparticles. Compatible with the usual technological processes such as soft lithography, cPDMS can be microstructured in a large range of thicknesses and geometries. Moreover, cPDMS can be quickly, irreversibly and perfectly sealed to glass, PDMS and silicon substrates, something that is not possible for conventional metallic electrodes. The first part of the manuscript is centered on the characterization of this material. Its electrical and surface properties that may turn out critical for microfluidic applications have been particularly studied. Electric fields present many opportunities to perform key functions in microfluidic (detection, separation, fluid or particles handling). We have chosen to assess the potential of cPDMS electrodes for two kinds of applications. Aspects of detection were first demonstrated using cyclic voltammetry measurements. This electrochemical method has enabled both to characterize the cPDMS surface while validating its potential as an electrochemical analysis tool. In the last part of this manuscript, cPDMS was tested for the electrokinetic manipulation of particles through thre study of different electrical fields with induced phenomena. This has led to the development of microfluidic devices (integrating cPDMS electrodes) designed for cell lysis and cells electrofusion.

Sepsis, commonly referred to as blood poisoning, is a serious medical condition characterized by a whole-body inflammatory state caused by microbial infection. Rapid treatment is crucial, however, traditional culture-based diagnostics usually takes 2-5 days.  The overall aim of the thesis is to develop microfluidic based sample preparation strategies, capable of isolating bacteria from whole blood for rapid sepsis diagnostics.  Although emerging technologies, such as microfluidics and “lab-on-a-chip” (LOC) devices have the potential to spur the development of protocols and affordable instruments, most often sample preparation is performed manually with procedures that involve handling steps prone to introducing artifacts, require skilled technicians and well-equipped, expensive laboratories.  Here, we propose the development of methods for fast and efficient sample preparation that can isolate bacteria from whole blood by using microfluidic techniques with potential to be incorporated in LOC systems. We have developed two means for high throughput bacteria isolation: size based sorting and selective lysis of blood cells. To process the large blood samples needed in sepsis diagnostics, we introduce novel manufacturing techniques that enable scalable parallelization for increased throughput in miniaturized devices. The novel manufacturing technique uses a flexible transfer carrier sheet, water-dissolvable release material, poly(vinyl alcohol), and a controlled polymerization inhibitor to enable highly complex polydimethylsiloxane (PDMS) structures containing thin membranes and 3D fluidic networks. The size based sorting utilizes inertial microfluidics, a novel particles focusing method that operates at extremely high flow rates. Inertial focusing in flow through a single inlet and two outlet, scalable parallel channel devices, was demonstrated with filtration efficiency of >95% and a flowrate of 3.2 mL/min. Finally, we have developed a novel microfluidic based sample preparation strategy to continuously isolate bacteria from whole blood for downstream analysis. The method takes advantage of the fact that bacteria cells have a rigid cell wall protecting the cell, while blood cells are much more susceptible to chemical lysis. Whole blood is continuously mixed with saponin for primary lysis, followed by osmotic shock in water. We obtained complete lysis of all blood cells, while more than 80% of the bacteria were readily recovered for downstream processing. Altogether, we have provided new bacteria isolation methods, and improved the manufacturing techniques and microfluidic components that, combined offer the potential for affordable and effective sample preparation for subsequent pathogen identification, all in an automated LOC format. / QC 20120611

This thesis reports the viability of E. coli cell extracts prepared using equipment that is both common to biotechnology laboratories and able to process small volume samples and expression of proteins containing unnatural amino acids (UAAs) at higher level using PCR amplified linear DNA templates (LETs) in cell-free protein synthesis (CFPS) system. E. coli-based cell extracts are a vital component of inexpensive and high-yielding CFPS reactions. However, effective preparation of E. coli cell extract is limited to high-pressure homogenizers (French press style or impinge-style) or bead mill homogenizers, which all require a significant capital investment. This work specifically assessed the following capital cost lysis techniques: (1) sonication, (2) bead vortex mixing, (3) freeze-thaw cycling, and (4) lysozyme incubation to prepare E. coli cell extract for CFPS. In this work, simple shake flask fermentation with a commercially available E. coli strain was used. Additionally, the RNA polymerase was over expressed in the E. coli cells prior to lysis which eliminated the need to add independently purified RNA polymerase to the CFPS reaction. As a result, high yielding E. coli-based cell extract was prepared using equipment requiring reduced capital investment and common to biotechnology laboratories. To our knowledge, this is the first successful prokaryote-based CFPS reaction to be carried out with extract prepared by sonication or bead vortex mixing. LETs are an attractive alternative to plasmids for site-specific incorporation of unnatural amino acids in proteins in the CFPS system because of their short preparation time and ease of production. However, major limitations associated with LETs are: (1) their degradation by RecBCD enzyme present in the cell-extract used for CFPS and (2) high CFPS energy costs. In this work, we report the optimization of LET-based CFPS for improved protein yield by inhibiting the RecBCD enzyme with small inhibitor molecules resulting in three fold increment in yield of protein containing UAA. We also assessed alternative energy sources such as glucose, fructose-1,6-bisphospate, creatine phosphate/creatine kinase, and high glutamate salt for cost reduction. This work could be important for high-throughput applications based on linear expression templates. This work demonstrates simple E. coli extract preparation and improved yield with linear expression templates for further advancements of cell-free protein synthesis system.

Pore forming toxins are known for their ability to efficiently form transmembrane pores which eventually leads to cell lysis. PFTs have potential applications in devel-oping novel drug and gene delivery strategies. Although structural aspects of many pore forming toxins have been studied, very little is known about the dynamics and subsequent rupture mechanisms. In the first part of the thesis, a combined experimental and modeling study to understand the lytic action of Cytolysin A (ClyA) toxins on red blood cells (RBCs) has been presented. Lysis experiments are carried out on a 1% suspension of RBCs for different initial toxin concentrations ranging from 100 – 500 ng/ml and the extent of lysis is monitored spectrophotometrically. Using a mean field approach, we propose a non – equilibrium adsorption-reaction model to quantify the rate of pore formation on the cell surface. By analysing the model in a pre-lysis regime, the number of pores per RBC to initiate rupture was found to lie between 400 and 800. The time constants for pore formation are estimated to lie between 1-25 s and monomer conformation time scales were found to be 2-4 times greater than the oligomerization times. Using this model, we are able to predict the extent of cell lysis as a function of the initial toxin concentration. Various kinetic models for oligomerization mechanism have been explored. Irreversible sequential kinetic model has the best agreement with the available experimental data. Subsequent to the mean field approach, a population balance model was also formulated. The mechanics of cell rupture due to pore formation is poorly understood. Efforts to address this issue are concerned with understanding the changes in the membrane mechanical properties such as the modulus and tension in the presence of pores. The second part of the thesis is concerned with using atomic force microscopy to measure the mechanical properties of cells. We explore the possibility of employing tapping mode AFM (TM-AFM) to obtain the elastic modulus of soft samples. The dynamics of TM-AFM is modelled to predict the elastic modulus of soft samples, and predict optimal cantilever stiffness for soft biological samples. From experiments using TM-AFM on Nylon-6,6 the elastic modulus is predicted to lie between 2 and 5 GPa. For materials having elastic moduli in the range of 1– 20 GPa, the cantilever stiffness from simulations is found to lie in the range of 1 – 50 N/m. For soft biological samples, whose elastic moduli are in the range of 10-1000 kPa, a narrower range of cantilever stiffness (0.1 – 0.6 N/m), should be used.

<p>Microbiological contamination from bacteria such as <em>Escherichia coli</em> and Salmonella is one of the main reasons for waterborne illness. Real time and accurate monitoring of water is needed in order to alleviate this human health concern. Performing multiple and parallel analysis of biomarkers such as DNA and mRNA that targets different regions of pathogen functionality provides a complete picture of its presence and viability in the shortest possible time. These biomarkers are present inside the cell and need to be extracted for analysis and detection. Hence, lysis of these pathogenic bacteria is an important part in the sample preparation for rapid detection. In addition, collecting a small amount of bacteria present in a large volume of sample and concentrating them before lysing is important as it facilitates the downstream assay. Various techniques, categorized as mechanical, chemical, thermal and electrical, have been used for lysing cells. In the electrical method, cells are lysed by exposure to an external electric field. The advantage of this method, in contrast to other methods, is that it allows lysis without the introduction of any chemical and biological reagents and permits rapid recovery of intercellular organelles. Despite the advantages, issues such as high voltage requirement, bubble generation and Joule heating are associated with the electrical method.</p> <p>To alleviate the issues associated with electrical lysis, a new design and associated fabrication process for a microfluidic cell lysis device is described in this thesis. The device consists of a nanoporous polycarbonate (PCTE) membrane sandwiched between two PDMS microchannels with electrodes embedded at the reservoirs of the microchannels. Microcontact printing is used to attach this PCTE membrane with PDMS.</p> <p>By using this PCTE membrane, it was possible to intensify the electric field at the interface of two channels while maintaining it low in the other sections of the device. Furthermore, the device also allowed electrophoretic trapping of cells before lysis at a lower applied potential. For instance, it could trap bacteria such as <em>E. coli</em> from a continuous flow into the intersection between two channels for lower electric field (308 V/cm) and lyse the cell when electric field was increased more than 1000 V/cm into that section.</p> <p>Application of lower DC voltage with pressure driven flow alleviated adverse effect from Joule heating. Moreover, gas evolution and bubble generation was not observed during the operation of this device.</p> <p>Accumulation and lysis of bacteria were studied under a fluorescence microscope and quantified by using intensity measurement. To observe the accumulation and lysis, LIVE/DEAD BacLight Bacterial Viability Kit consisting of two separate components of SYTO 9 and propidium iodide (PI) into the cell suspension in addition to GFP expressed <em>E. coli</em> were used. Finally, plate counting was done to determine the efficiency of the device and it was observed that the device could lyse 90% of bacteria for an operation voltage of 300V within 3 min.</p> <p>In conclusion, a robust, reliable and flexible microfluidic cell lysis device was proposed and analyzed which is useful for sample pretreatment in a Micro Total Analysis System.</p> / Master of Applied Science (MASc)