Degree Type

Dissertation

Date of Award

2021

Degree Name

Doctor of Philosophy

Department

Chemistry

Major

Analytical Chemistry

First Advisor

Jared L. Anderson

Abstract

Sample preparation is vital in nucleic acid analysis. Cell lysis is generally the first step in DNA sample preparation where the cell membrane is disrupted to release intracellular components such as DNA. Moreover, the isolation and enrichment of nucleic acids is essential to prevent false negative results from enzymatic detection methods that are sensitive to impurities. Conventional nucleic acid sample preparation methods are tedious and time-consuming, limiting sample throughputs and the ability for automation and point-of-care diagnostics. Given the drawbacks of conventional methods and the need to provide rapid results, innovative cell lysis and DNA purification procedures should be investigated to improve sample throughputs while ensuring sample purity.Magnetic ionic liquids (MILs) are molten salts that exhibit magnetic susceptibility due to a paramagnetic component within the chemical structure of the anion or cation. As a subclass of ionic liquids (ILs), MILs exhibit similar advantageous physicochemical properties to ILs, such as negligible vapor pressure and tunable viscosity. The paramagnetic nature of MILs has attracted substantial interest in sample preparation technologies as the solvent can be rapidly collected using a magnet avoiding traditional centrifugation steps required to collect the analyte-enriched extraction solvent. Nucleic acid analysis can be broken into three distant steps, including 1) cell lysis, 2) DNA extraction, and 3) detection. DNA sample preparation is essential to remove cellular debris and impurities to permit enzymatic detection (i.e., polymerase chain reaction (PCR) and sequencing). Therefore, hexafluoroacetylactonate-based MILs were investigated as DNA extraction solvents to preconcentrate nucleic acids from complex matrices such as plasma, blood, and artificial sputum. The DNA-enriched MIL was collected on a rod magnet and directly integrated into quantitative PCR (qPCR) and multiplex-qPCR assays with SYBR green and Taqman probe detection. Adding the DNA-enriched MIL into the reaction buffer drastically reduced the sample preparation time improving sample throughputs without inhibiting PCR efficiency. A sequence-specific preconcentration step may be required to prevent low abundance nucleic acids from being masked by background DNA. Wild-type DNA sometimes differs by a single nucleotide, making it challenging to preconcentrate only the mutant target. Therefore, ion-tagged oligonucleotides (ITOs) were previously designed to capitalize on Watson-Crick base pairing to selectively anneal to DNA sequences. An ITO probe contains an imidazolium-based tag that allows the probe to be captured by a hydrophobic MIL that poorly extracts nucleic acids. A rapid sequence-specific DNA extraction (i.e., 11 min) to preconcentrate target nucleic acids was developed by dispersing a manganese(II)-based MIL in the sample after the ITO probe annealed to the target DNA. The DNA-enriched MIL was then integrated into the qPCR assay for analysis. The dispersive ITO-MIL sequence-specific extraction method could selectively extract nucleic acids from plasma, artificial sputum, and artificial urine. With the ITO-MIL extraction, 0.1% BRAF V600E (99.9% wild-type BRAF) could be discriminated from a 100% wild-type BRAF standard. In comparison, without the sequence-specific extraction, the 9% BRAF V600E could not be differentiated from the 100% wild-type BRAF standard. Commercial streptavidin-coated magnetic beads with biotin-modified oligonucleotides were unable to selectively extract target nucleic acids from plasma. This suggests that a total DNA extraction should be performed prior to a sequence-specific extraction using the commercial beads, which would drastically limit sample throughputs.

Cell lysis is the vital first step in DNA analysis. The cell membrane is either solubilized by a lysis reagent or disrupted by physical stress. However, conventional chemical cell lysis methods often inhibit downstream bioanalytical detection requiring substantial purification. In comparison, mechanical lysis methods need to be strong enough to break the cells. However, if the shear and friction forces are too strong genomic DNA can be damaged. To overcome the limitations of conventional cell lysis methods, MILs were investigated as lysis reagents. A hydrophobic MIL was dispersed in a blood sample and recovered with a magnet. The MIL simultaneously lysed blood cells and extracted DNA during the dispersion step. The 1 min sample preparation method using MILs captured picogram levels of genomic DNA without inhibiting the qPCR reaction. The metal ion incorporated within the MIL (i.e., Ni(II), Co(II), Dy(III), and Gd(III)) appears to cause hemolysis, while the cationic component reduces the cell’s integrity by interacting with the cell membrane.

DOI

https://doi.org/10.31274/etd-20210609-50

Copyright Owner

Miranda N. Emaus

Language

en

File Format

application/pdf

File Size

198 pages

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