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Full-Text Articles in Physical Sciences and Mathematics
Molecular-Level Studies Of Nanopatterned Biomolecules With Atomic Force Microscopy, Ashley R. Walker
Molecular-Level Studies Of Nanopatterned Biomolecules With Atomic Force Microscopy, Ashley R. Walker
LSU Doctoral Dissertations
Atomic force microscopy (AFM) is an analytical technique in which a tipped probe is gently scanned across the surface in a raster pattern to generate digital images of a sample at the nanoscale. The AFM instrument has three general operational modes, which are contact, non-contact and tapping-mode, that can be used to examine materials at the atomic level. Single-molecular details of biological molecules and other soft organic materials can be captured with minimal denaturation in either ambient or liquid environments when using tapping-mode AFM. In tapping-mode, the probe is driven to oscillate vertically while the tip is scanned across the …
Infrared Laser Ablation For Biomolecule Sampling, Kelin Wang
Infrared Laser Ablation For Biomolecule Sampling, Kelin Wang
LSU Doctoral Dissertations
In this research, an infrared laser at a wavelength of 3 µm was used to ablate material from tissue sections for biomolecule analysis. Pulsed infrared (IR) irradiation of tissue with a focused laser beam efficiently removed biomolecules, such as proteins, enzymes, DNA, and RNA from tissue sections for further analysis. In a proteomics project, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to determine regions of interest (ROI) for laser ablation. The matrix was then washed off. By overlaying the MSI generated heat-map, the section was sampled using IR laser ablation and custom stage-control software. Two ROI were selected …
Atomic Force Microscopy Tip-Enhanced Laser Ablation, Fan Cao
Atomic Force Microscopy Tip-Enhanced Laser Ablation, Fan Cao
LSU Doctoral Dissertations
In the present work, an apertureless atomic force microscope (AFM) tip-enhanced laser ablation (TELA) system was developed and investigated. An AFM was coupled to an optical parametric oscillator (OPO) wavelength tunable laser for sample ablation with a submicron sampling size. The AFM was used to image the surface and hold the AFM tip 10 nm above the sample surface. The AFM tip is coated with a layer of gold with a thickness of 35 nm. The incident laser wavelength was tuned in the visible and near-infrared (IR) region and focused on the AFM tip. With the tip-enhancement effect, ablation craters …
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.
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 …