Biomolecules in Nanoporous Structures

• Author / Creator

  Zhou, Ya

• Nanomaterials have been widely involved in biomolecular analysis, to refine traditional analytical method or develop complementary techniques. In this thesis, nanoporous structures were utilized in two applications, DNA electrophoresis and solid matrix laser desorption ionization (SMALDI). Microfabricated devices utilizing ratchet based motion of DNA may offer a far more rapid method for large DNA fragment separation, useful in genetic studies. However, on-chip separation of megabase-sized DNA in nanoporous colloidal self-assembled (CSA) structures within microfluidic devices is limited by DNA trapping, a phenomenon that also limits the speed and efficiency of gel electrophoresis. Megabase-sized DNA is crucial to modern genomics research of all organisms. In Chapter 2, we investigated the mechanism of DNA trapping in silica nanoporous structure. We found the primary reason for DNA trapping is the reduced mobility of apex segments of U/J shaped DNA. The pinning of the apex and the thriving of near-apex hernias result in an irreversible trapping conformation of DNA molecule in the separation sieves. Insulator-based dielectrophoresis (DEP) origins from the structure of the separation sieves and is proposed to be the source of the external force on apex segments. The presence of DEP during DNA electrophoretic separation and its effects are often neglected since DEP force is considerably weaker than electrophoretic force. However, we found the perturbation by DEP force at apex confounds the Ratchet model which regulates the DNA motion and separation mechanism by pulsed-field electrophoresis. This clear trapping mechanism contrasts with those for trapping in gels, which give no clear explanation for a strong field dependence. Using reverse or intermittent field spikes greatly increases the field strength available, allowing much more rapid separations than in gels. In another work, porous silicon thin film fabricated by glancing angle deposition (GLAD) was utilized for the solid matrix laser desorption ionization (SMALDI). Not involving the organic matrix in the laser desorption ionization (LDI) process, SMALDI is suitable for analyzing small molecules, particularly metabolites, with a simple spot test. However, salt and other sample matrix components in biofluids greatly reduce the performance of SMALDI as a metabolite assay. Herein, we describe a unique approach to surface modification that gives an on-line sample preparation method for SMALDI and possibly other porous silicon based LDI techniques. The approach separates ionic metabolites, such as amino acids, and dominant background electrolytes, which are usually so similar in solubility and hydrophobicity that they are not readily separated by a surface enhanced LDI approach, instead requiring the more laborious and method of LC-MS. The background electrolytes are directly segregated through crystallization on perfluoro coated SMALDI surface, which are prepared with coating defects to cause the salt segregation during spot drying. Combined with desalting, the quantitative analysis of metabolites in salty samples (serum and artificial cerebrospinal fluid) by SMALDI-MS is improved, and the technique has potential to be applied to metabolite profiling in batches.

• Subjects / Keywords

  • Laser Desorption Ionization
  • Metabolite Desalting
  • DNA Trapping
  • Pulsed Field Electrophoresis

• Graduation date

  Spring 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R33X84164

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.