Synchronous Optical and Electrical Measurements of Single DNA Molecules Translocating Through a Solid-State Nanopore

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dc.contributor.authorBustamante, José
dc.date.accessioned2015-01-09T16:27:47Z
dc.date.available2015-12-15T09:00:07Z
dc.date.created2015
dc.date.issued2015
dc.identifier.urihttp://hdl.handle.net/10393/31903
dc.identifier.urihttp://dx.doi.org/10.20381/ruor-5739
dc.description.abstractNanopore sensors are emerging as a promising technology for single molecule analysis and polymer sequencing. Traditionally, measurements are taken by monitoring the ionic current through the nanopore, which gives information (e.g. size, shape, charge) about a molecule of interest while it is in the confined geometry of the nanopore. The dynamics of the molecule before the arrival to the nanopore, such as the capture dynamics, or molecular conformation prior to translocation, as well as clogging mechanisms and features of anomalous translocation events, are not assessed by the electrical measurements alone. To study the whole process of nanopore diffusion, capture and passage it is necessary to complement the electrical signal with another detection mode. Particularly, optical visualization of the molecules as they translocate through the nanopore has great potential. In this Thesis I present the design, construction, optimization and testing of a nanopore--‐based optofluidic instrument, which uses fluorescence microscopy to visualize individual fluorescently stained DNA molecules as they translocate a solid--‐state nanopore, while in parallel record the ionic current signal through the pore. The following challenges were overcome to achieve the integration of the optical and electrical systems: (i) the electrical detection system must account for the physical constrains of a wide field fluorescence microscope, and the optical system should in turn not affect the low--‐noise electrical detection of individual DNA molecules. The design of the instrument included a microfluidic device, so to position the nanopore within the working distance (<170--‐μm) of the microscope objective (Chapter 2). (ii) Electrical noise was optimized to a level that is indistinguishable from a standard (with no optics) nanopore system (Chapter 3). The custom instrument was used to demonstrate: 1) Electrical detection of DNA translocations with a laser light illuminating the nanopore; 2) Optical tracking of DNA capture and translocation dynamics; 3) Synchronization of the optical and electrical signals in preparation for simultaneous detection. In the process of noise optimization, a strong noise coupling between the illumination source and the ionic current was found, characterized and eliminated. Consequently, the noise performance of the custom instrument is the lowest of any other nanopore--‐based optofluidic systems described in the literature to date. This opens up the way to many new and exciting investigations of polymer translocation dynamics through nanoconfined geometries. Lastly, during the development of this custom instrument, a method to localize the fabrication of a nanopore by controlled dielectric breakdown on a membrane, with a focused laser beam, was discovered.
dc.language.isoen
dc.publisherUniversité d'Ottawa / University of Ottawa
dc.subjectNanopore
dc.subjectOptical and Electrical DNA detection
dc.subjectNanopore-based Optofluidic System
dc.subjectDNA
dc.subjectFluorescence Microscopy
dc.subjectNoise Optimization
dc.titleSynchronous Optical and Electrical Measurements of Single DNA Molecules Translocating Through a Solid-State Nanopore
dc.typeThesis
dc.faculty.departmentPhysique / Physics
dc.contributor.supervisorTabard-Cossa, Vincent
dc.embargo.terms2015-12-15 00:00:00
dc.degree.nameMSc
dc.degree.levelmasters
dc.degree.disciplineSciences / Science
thesis.degree.nameMSc
thesis.degree.levelMasters
thesis.degree.disciplineSciences / Science
uottawa.departmentPhysique / Physics
CollectionThèses, 2011 - // Theses, 2011 -

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