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Dynamic Nanochannels for Biosensing ApplicationsOxborrow, Joseph B 01 November 2013 (has links) (PDF)
Inexpensive label-free detection of biomarker panels in serum could revolutionize earlycancer diagnosis and treatment. Such detection capabilities may be possible with dynamicnanochannels in conjunction with electrical impedance measurement. In Dr. Greg Nordin's lab I designed, fabricated and tested several iterations of these sensors with polydimethyl-siloxane microfluidics. The final design yielded a dynamic nanochannel array sensor thatshowed a 140% impedance change when exposed to 14µM bovine serum albumin in phos-phate buffered saline. For the geometry and noise limits of the tested device, simulationsindicated that a minimum detectable concentration of 20pM with specifically bound strep-tavidin should be possible. However, the polydimethylsiloxane approach is also shown to beproblematic in meeting the trade-offs required for a practical device. Consequently, alter-native materials and designs are suggested to reduce the minimum detectable concentrationto the high femtomolar range, which would be attractive for detection of many medicalbiomarkers.
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Fabrication and characterization of sub-micron and nanoscale structures in commercial polymersIbrahim, Fathima Shaida January 1900 (has links)
Doctor of Philosophy / Department of Chemistry / Takashi Ito / This dissertation describes the fabrication and characterization of nanoscale structures in commercially available polymers via multiphoton ablation and bottom-up self assembly techniques. High-resolution surface imaging techniques, such as atomic force microscopy (AFM) and chemical force microscopy (CFM) were used to characterize the physical features and chemical properties, respectively, of these nanoscale structures. Fabrication using both top-down and bottom-up methods affords flexibility in that top-down allows random, user-defined patterning whereas bottom-up self assembly produces truly nanoscale (1-100nm) uniform features. Multiphoton induced laser ablation, a top-down method, was used to produce random sub-micron scale features in films of poly(methylmethacrylate) (PMMA), polystyrene (PS), poly(butylmethacrylate) (PBMA) and poly[2-(3-thienyl)ethyloxy-4-butylsulfonate] (PTEBS). Features with 120-nm lateral resolution were obtained in a PMMA film which was concluded to be the best polymer for use with this method. It was also found that etching resolution was highest for polymers having high glass transition temperatures, low molecular weights and no visible absorption. Bottom-up self assembly of polystyrene-poly (methylmethacrylate) (PS-b-PMMA) diblock copolymer and UV/acetic acid treatment produced nanoscale cylindrical domains supported by a substrate. AFM imaging at the free surface showed metastable vertical PMMA domain orientation on gold substrates. In contrast, horizontal orientation was obtained on oxide-coated silicon regardless of surface roughness and annealing conditions. The horizontal domain orientation on silicon substrates was ideal to probe simultaneously the difference in surface charge and hydrophilicity of the two distinct nanoscale domains of UV/AcOH treated PS-b-PMMA films. CFM on UV/acetic acid etched PS-b-PMMA revealed the presence of –COO- groups which were found to be more abundant inside the etched trenches than on the unetched PS matrix as shown by ferritin adsorption onto etched PS-b-PMMA. Lastly, the PS-b-PMMA was cast as a free-standing monolith at the end of a quartz micropipette. AFM revealed circular PMMA dots at the free surface, indicating alignment parallel to the long axis of capillary. Ion conductance within nanochannels indicated surface –charge governed ion transport at low KCl concentrations and flux of negatively-charged sulphorhodamine dye demonstrated the permselective nature of nanochannels.
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Microfluidic Devices and BiosensorsTsai, Long-Fang 01 February 2016 (has links)
My research broadly covers various important aspects of microfluidic devices and biosensors. Specifically, this dissertation reports: (1) a new and effective room temperature method of bonding polydimethylsiloxane (PDMS) microfluidics to substrates such as silicon and glass, (2) a new microfluidic pump concept and implementation specifically designed to repeatedly drive a small sample volume (<1 µL) very rapidly (~500 µL/min) through a sensor-containing flow channel to significantly decrease sensor response time through advection-driven rather than diffusion-driven mass transport, (3) use of a new microfluidic material based on polyethylene glycol diacrylate (PEGDA) to implement impedance-based dynamic nanochannel sensors for protein sensing, and (4) an investigation of galvanoluminescence and how to avoid it for conditions important to fluorescence-based dielectrophoresis (DEP) microfluidic biosensors. Over the last decade, the Nordin research group has developed a lab-on-a-chip (LOC) biosensor based on silicon photonic microcantilever arrays integrated with polydimethylsiloxane (PDMS) microfluidics for protein biomarker detection. Integration requires reliable bonding at room temperature with adequate bond strength between the PDMS element and microcantilever sensor substrate. The requirement for a room temperature process is particularly critical because microcantilevers must be individually functionalized with antibody-based receptor molecules prior to bonding and cannot withstand significant heating after functionalization. I developed a new room temperature bonding method using PDMS curing agent as an intermediate adhesive layer. Two curing agents (Sylgard 184 and 182) were compared, as well as an alternate UV curable adhesive (NOA 75). The bond strength of Sylgard 184 was found to be stronger than Sylgard 182 under the same curing conditions. Overnight room temperature curing with Sylgard 184 yields an average burst pressure of 433 kPa, which is more than adequate for many PDMS sensor devices. In contrast, UV curable epoxy required a 12 hour bake at 50 °C to achieve maximum bond strength, which resulted in a burst pressure of only 124 kPa. In many biosensing scenarios it is desirable to use a small sample volume (<1 µL) to detect small analyte concentrations in as short a time as possible. I report a new microfluidic pump to address this need, which we call a reflow pump. It is designed to rapidly pump a small sample volume back and forth in a flow channel. Ultimately, the flow channel would contain functionalized sensor surfaces. The rapid flow permits use of advection-driven mass transport to the sensor surfaces to dramatically reduce sensor response times compared to diffusion-based mass transport. Normally such rapid flow would have the effect of decreasing the fraction of analyte molecules in the volume that would see the sensor surfaces. By configuring the pump to reflow fluid back and forth in the flow channel, the analyte molecules in the small sample volume are used efficiently in that they have many opportunities to make it to the sensor surfaces. I describe a 3-layer PDMS reflow pump that pumps 300 nL of fluid at 500 µL/min for 15 psi actuation pressure, and demonstrate a new two-layer configuration that significantly simplifies pump fabrication. Impedance-based nanochannel sensors operate on the basis of capturing target molecules in nanochannels such that impedance through the nanochannels is increased. While simple in concept, the response time can be quite long (8~12 hours) because the achievable flow rate through a nanochannel is very limited. An approach to dramatically increase the flow rate is to form nanochannels only during impedance measurements, and otherwise have an array of nanotrenches on the surface of a conventional microfluidic flow channel where they are exposed to normal microfluidic flow rates. I have implemented such a dynamic nanochannel approach with a recently-developed microfluidic material based polyethylene glycol diacrylate (PEGDA). I present the design, fabrication, and testing of PEGDA dynamic nanochannel array sensors, and demonstrate an 11.2 % increase in nanochannel impedance when exposed to 7.2 µM bovine serum albumin (BSA) in phosphate buffered saline (PBS). Recently, LOC biosensors for cancer cell detection have been demonstrated based on a combination of dielectrophoresis (DEP) and fluorescence detection. For fluorescence detection it is critical to minimize other sources of light in the system. However, reported devices use a non-noble metal electrode, indium tin oxide (ITO), to take advantage of its optical transparency. Unfortunately, use of non-noble metal electrodes can result in galvanoluminescence (GL) in which the AC voltage applied to the electrodes to achieve DEP causes light emission, which can potentially confound the fluorescence measurement. I designed and fabricated two types of devices to examine and identify conditions that lead to GL. Based on my observations, I have developed a method to avoid GL that involves measuring the impedance spectrum of a DEP device and choosing an operating frequency in the resistive portion of the spectrum. I also measure the emission spectrum of twelve salt solutions, all of which exhibited broadband GL. Finally, I show that in addition to Au, Cr and Ni do not exhibit GL, are therefore potentially attractive as low cost DEP electrode materials.
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Poly(dimethylsiloxane) Based Micro- and Nanofluidic Device Fabrication for Electrophoresis ApplicationsPussadee, Nirut 04 November 2010 (has links)
No description available.
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Towards genome-wide, single-molecule analysis of eukaryotic DNA replication / Vers l'analyse en molécule unique de la réplication de l'ADN eucaryote à l'échelle du génome entierDe Carli, Francesco 28 September 2016 (has links)
Chez les eucaryotes, la réplication de l'ADN démarre au niveau de multiples origines activées suivant un programme précis, qui peut être analysé à l'échelle du génome sur des populations cellulaires. Cependant, l'étude de la variabilité intercellulaire, la détection d'évènements rares et la mesure de la vitesse des fourches de réplication nécessitent des analyses en molécule unique. Avec les techniques actuelles, l'ADN néosynthétisé est marqué avec des analogues de la thymidine et révélé par des anticorps fluorescents. Les molécules d'intérêt sont identifiées par hybridation fluorescente in situ. Ces étapes sont complexes et le débit est faible. Cette thèse développe de nouvelles méthodes de détection et d'identification des molécules d'ADN réplicatives sans anticorps et à haut débit. L'ADN est répliqué en présence d'un dUTP fluorescent, purifié puis marqué en code-barre spécifique permettant l'alignement sur le génome de référence par coupure avec une endonucléase simple brin et incorporation d'un autre dUTP fluorescent. L'ADN est ensuite coloré avec un intercalant fluorescent, le YOYO-1. Les molécules d'ADN, leurs segments néorépliqués et leurs code-barres sont observés en trois couleurs différentes par épifluorescence directe. Les segments répliqués ont une fluorescence YOYO-1 plus intense, ce qui permet de détecter les bulles de réplication sans marquage métabolique. Ces outils ont été couplés à un dispositif nanofluidique dans lequel l'ADN est conduit dans des milliers de nanocanaux et imagé automatiquement, ce qui augmente massivement le débit. L'ensemble de ces résultats ouvre la voie à la cartographie pangénomique de la réplication de l'ADN en molécule unique. / In eukaryotes, DNA replication starts at multiple origins that are activated following a specific program. Population methods allow genome-wide analysis of DNA replication. However, single-molecule methods are required to monitor cell-to-cell variability, detect rare events and measure individual replication fork speeds. With the existing techniques, newly-synthesized DNA is labelled with thymidine analogs and revealed with fluorescent antibodies. Fibres containing a locus of interest can be identified by fluorescent in situ hybridization. These steps are complex and the throughput is low. This work proposes novel, antibody-free tools to detect replication tracts and identify the locus of origin of all DNA molecules at much higher throughput. DNA replicated in the presence of a fluorescent dUTP was purified and specifically barcoded by using a nicking endonuclease, followed by limited nick-translation with another fluorescent dUTP. This allowed alignment to a reference genome map. DNA was then stained with the fluorescent DNA intercalator YOYO-1. Direct epifluorescence revealed the DNA molecules, their replication tracts and their barcodes in three distinct colours. Replicated segments showed a stronger YOYO-1 fluorescence, demonstrating that replication bubbles can be directly detected without metabolic labelling. Finally, these tools were coupled to a nanofluidic device: DNA was driven into 13,000 parallel nanochannels and automatically imaged, massively increasing the throughput. Altogether, these results provide a starting point for genome-wide, single-molecule mapping of DNA replication in eukaryotic organisms.
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Electrokinetic flow in micro- and nano-fluidic componentsZheng, Zhi 19 November 2003 (has links)
No description available.
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