Fundamentals and applications of solid state and biological nanopores
Creators: name: Perera, Rukshan Tharanga
Publisher: name: University of Utah
Year: 2016
Subjects: Activation energy
Alpha hemolysin
Benzo[a]pyrene
Biomarkers
DNA
RNA
PNA unzipping
Ion transport

description: Both biological and solid state nanopores have been used to study fundamentals of ion transport phenomena and potential applications in single molecule analysis. This dissertation presents investigations of the effect of the electric double layer in the activation energy of ion transport in conical shaped glass nanopores. Further, it presents the use of the a-hemolysin nanopore to detect the DNA cancer biomarker benzo[a]pyrene, and to detect structural differences between the A- and B- form duplexes. Chapter 1 overviews the solid state and biological nanopores, common transport phenomena observed in sold state nanopores, and detection of ssDNA, dsDNA, and biomarkers using biological nanopores. Chapter 2 describes the effect of the electric double layer on the activation energy of ion transport through conical shaped glass nanopores. The study shows that the activation energy values for transport within an electrically charged conical glass nanopore differ from the bulk values due to the voltage and temperature-dependent distribution of the ions within the double layer. Finite element simulations based on the Poisson-Nernst- Planck model semiquantitatively predict the measured temperature-dependent conductivity and dependence of activation energy (Ea) on applied voltage. The results highlight the relationships between the distribution of ions with the nanopore, ionic current, and E a, and their dependence on pore size, temperature, ion concentration, and applied voltage. Chapter 3 describes how the a-hemolysin (aHL) nanopore platform can be used to detect the benzo[a]pyrene diol epoxide (BPDE) adduct to guanine (G) in synthetic oligo deoxynucleotides. BPDE adducts are formed by exposure to the carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, and considered as a biomarker that can initiate cancers. Translocation of a 41-mer poly-2'-deoxycytidine strand with a centrally located BPDE adduct to G through aHL in 1 M KCl produces a unique multilevel current signature allowing the adduct to be detected from either the 5' or 3' directions. This result suggests that BPDE adducts and other large aromatic biomarkers can be detected with aHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples. Chapter 4 describes the unzipping of double-stranded nucleic acids by an electric field applied across the membrane, providing structural information about different duplex forms. Comparative studies on A-form DNA-RNA duplexes and B-form DNA-DNA duplexes with a single-stranded tail identified significant differences in the blockage current and in the unzipping duration between the two helical forms. The effect of varying the length of the single-stranded overhang was investigated, and A-form DNA-PNA duplexes were studied to provide additional support for the proposed model. This result identifies key differences between A- and B-form duplex unzipping that will be important in the design of future probe-based methods for detecting DNA or RNA.