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Full-Text Articles in Physical Sciences and Mathematics
Single Molecule Electrophoresis And Optical Detection Using Thermoplastic Nanofluidic Devices: An Experimental And Simulation Study, Kumuditha Madushanka Weerakoon Ratnayake
Single Molecule Electrophoresis And Optical Detection Using Thermoplastic Nanofluidic Devices: An Experimental And Simulation Study, Kumuditha Madushanka Weerakoon Ratnayake
LSU Doctoral Dissertations
Nanofludic devices provide a great platform for single molecular analysis. The unique phenomena in nanoscale gained such interest in investigating the single molecular behavior in nanochannels. Sizes less than 200 nm in one or two-dimensional structures have lead to fascinating observations not accessible in microscale. When a single molecule translocates through a nanotube it interacts with channel walls by adsorption/ desorption, van der Waals interactions and hydrophilic interactions providing a mechanism for separation without any extra additives. Moreover, double layer thickness governed by the background electrolyte plays a vital role. We report single molecular electrophoresis phenomena in nanochannels and nanoslits …
Microfluidics For The Analysis Of Integral Membrane Proteins: A Top-Down Approach, Katrina Nychole Battle
Microfluidics For The Analysis Of Integral Membrane Proteins: A Top-Down Approach, Katrina Nychole Battle
LSU Doctoral Dissertations
The development of fully automated and high-throughput systems for proteomics is now in demand because of the need to generate new protein-based disease biomarkers. Unfortunately, it is difficult to identify protein biomarkers that are low abundant when in the presence of highly abundant proteins, especially in complex biological samples like serum, cell lysates, and other biological fluids. Membrane proteins, which are in many cases of low abundance compared to cytosolic proteins, have various functions and can provide insight into the state of disease and serve as targets for new drugs making them attractive biomarker candidates. Traditionally, proteins are identified through …
Revolutionizing Genomic Analyses: Mutation Analyses Using Novel Enzyme-Based Assays With Laser-Induced Fluorescence And Polymeric Microfluidic Devices As Electrophoretic Platforms, Rondedrick Deshaun Sinville
Revolutionizing Genomic Analyses: Mutation Analyses Using Novel Enzyme-Based Assays With Laser-Induced Fluorescence And Polymeric Microfluidic Devices As Electrophoretic Platforms, Rondedrick Deshaun Sinville
LSU Doctoral Dissertations
Polymer-based microelectrophoresis was investigated to analyze known (mutation detection) and unknown (mutation scanning) low-abundant mutations in genomic DNA with high diagnostic value for colorectal cancers. For our mutation detection assays, point mutations in the K-ras oncogene were identified using the ligase detection reaction (LDR). For the mutation scanning assay, which searches for sporadic mutations, an EndoV-LDR assay was utilized with mutations in the p53 tumor suppressor gene used as a model. A poly(methylmethacrylate), PMMA, microchip filled with a 4% linear polyacrylamide (LPA) gel was used to electrophoretically sort products formed from LDRs, which produced oligonucleotides <65 bp in length. Using microchip electrophoresis with the LPA, a 44 bp ligation product was resolved from a 100-fold molar excess of unligated primers (25 bp) in approximately 120 s, which was ~17 times faster than conventional capillary gel electrophoresis. In order to simplify the electrophoretic process and further reduce development time, the LDR products were sorted in the absence of the sieving gel using free solution conjugate electrophoresis (FSCE). FSCE incorporated polyamide “drag-tags” onto LDR primers, which provided DNA fragment mobilities in free solution that were dependent upon their size. LDR/drag-tagged (LDR-dt) products could be formed in a multiplexed format for mutant-to-wild-type ratios as low as 1 to 100 with single base resolution. Separations were conducted using capillary array electrophoresis (CAE) and PMMA microchips filled with only a TRIS buffer. Analysis times for the LDR-dt products were less than 11 min using CAE and ~85 s for PMMA microchips with high reproducible migration times within and between microchips. PMMA-based microchips were also evaluated for the identification of sporadic mutations using an endonuclease V – LDR (Endo V/LDR) technique. Endo V cleaves heteroduplexed DNA one base 3’ of single-base mismatched sites as well as nicking DNA at some matched sites as LDR reseals miscleaved sites to reduce false positive signals. Results suggested that Endo V/LDR products from p53 mutations could be successfully separated and detected using a PMMA microfluidic chip filled with a sparsely cross-linked replaceable polyacrylamide gel in less than 6 min, which was approximately 10-fold shorter compared to CAE.
Application Of Polymeric Microfluidic Devices For Separation Of Single-Stranded Dna, Shawn Llopis
Application Of Polymeric Microfluidic Devices For Separation Of Single-Stranded Dna, Shawn Llopis
LSU Doctoral Dissertations
Microsystems targeted for DNA sequencing, especially those focused on electrophoretic separations, are rapidly proving their viability to genomic research, mimicking the progress made when capillary electrophoresis developed from miniaturizing slab gel electrophoresis techniques. Being the more recent electrophoretic separation platform, the commercial availability of microchip electrophoresis devices remains relatively limited. To this extent, high-aspect ratio microstructures formed in thermo plastics have been developed using rapid fabrication methods from molding tools designed for mass replication of high-aspect ratio microfeatures. In this work, the choice and compatibility of poly(methylmethacrylate) (PMMA) – the primary substrate for DNA separations in this work – was …
Fabricating Microfluidic Devices In Polymers For Bioanalytica Applications, Sean M. Ford
Fabricating Microfluidic Devices In Polymers For Bioanalytica Applications, Sean M. Ford
LSU Doctoral Dissertations
The research presented in this document focuses on the fabrication, characterization and application of microfluidic systems fabricated in poly(methyl methacrylate) (PMMA) with the emphasis focused on the fabrication processing steps. Microfluidic devices were produced in PMMA using X-ray lithography. The fabrication methods investigated were sacrificial mask, polyimide membrane mask and embossing techniques. PMMA microfluidic devices fabricated using X-ray lithography were characterized using scanning electron microscopy (SEM) and optical microscopy, while analytical techniques such as electroosmotic flow determination, separations, and fluorescent microscopy were used to characterize fluid transport in these devices. A novel method for the heat annealing of PMMA to …
Analysis Of Near-Infrared Dye-Labeled Sanger Sequencing Fragments With Gel Electrophoresis Using The Time-Resolved Flourescence Lifetime Indentification Methods, Suzanne Jeanel Lassiter
Analysis Of Near-Infrared Dye-Labeled Sanger Sequencing Fragments With Gel Electrophoresis Using The Time-Resolved Flourescence Lifetime Indentification Methods, Suzanne Jeanel Lassiter
LSU Doctoral Dissertations
The research presented in this dissertation involves the identification of sequencing fragments with time-resolved methods. For this application, near-infrared heavy-atom tricarbocyanine dyes were developed in our laboratory, which can be excited with a single laser and emission collected using a single detection channel. The dyes have four spectroscopically unique, but relatively short lifetimes that can be altered by the intramolecular heavy-atom they contain. The work described here involves the optimization of dye-primer chemistry for preparing Sanger sequencing reactions for longer reads and the optimization of the separation matrix for capillary gel electrophoresis that produces favorable statistical analysis of the aforementioned …