The thickness shear mode acoustic wave (TSM) sensor, operated in a flow-through format, has been widely used in bioanalytical research. My research is mainly focused on the study of surface-attached biomolecules and cells using the TSM sensor, including lesions in DNA, conformational change of calmodulin, as well as the properties and attachment of rat aortic smooth muscle cells.
Aldehydic apurinic or apyrimidinic sites (AP sites) that lack a nucleobase moiety are one of the most common forms of toxic lesions in DNA. In this work, synthesized oligodeoxyribonucleotides containing one, two, or three abasic sites were hybridized to complementary sequences immobilized on the gold electrode of the TSM device by affinity binding. The influence of AP sites on local base stacking energy and geometry caused a dramatic destabilization of the DNA duplex structure, which was detected by the TSM sensor. The signals detected by TSM correlated well with the thermostability of DNA duplexes in solution. The results indicate that both the number of sites and their localization in the double-stranded structure influence the stability of a 19 b.p. duplex.
TSM was also used to detect the binding of ions or peptides to surface-attached calmodulin. The interaction between calmodulin and ions induced an increase in resonant frequency and a decrease in motional resistance. In addition, these signal changes were reversible upon washing with buffer. The response was interpreted as a decrease in surface coupling induced by exposure of hydrophobic domains on the protein, and an increase in the length of calmodulin by approximately 3 Å. In addition, the interaction of the protein with peptide together with calcium ions was detected successfully, despite the relatively low molecular mass of the 2-kDa peptide.
In addition, the attachment of smooth muscle cells to various surfaces has been monitored by TSM. These surfaces include laminin, fibronectin and bare gold. The results of these experiments in terms of changes of frequency (fs) and resistance (Rm) were analyzed. The responses of the surface-bound cells to the introduction of various ions, depolarisation events and damage subsequent to exposure to hydrogen peroxide were also observed. Morphological changes in the cells, as confirmed by atomic force microscopy and scanning electron microscopy, are correlated with results from the TSM sensor.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/17248 |
| Date | 26 February 2009 |
| Creators | Wang, Xiaomeng |
| Contributors | Thompson, Michael |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
| Format | 2658899 bytes, application/pdf |
Page generated in 0.0064 seconds