Real-time monitoring and manipulation of single bio-molecules in free solution

dc.contributor.advisor Edward S. Yeung
dc.contributor.author Li, Hung-Wing
dc.contributor.department Chemistry
dc.date 2018-08-25T04:37:52.000
dc.date.accessioned 2020-06-30T07:46:47Z
dc.date.available 2020-06-30T07:46:47Z
dc.date.copyright Sat Jan 01 00:00:00 UTC 2005
dc.date.issued 2005-01-01
dc.description.abstract <p>The observation and manipulation of single biomolecules allow their dynamic behaviors to be studied to provide insight into molecular genetics, biochip assembly, biosensor design, DNA biophysics.;In a PDMS/glass microchannel, a nonuniform electroosmotic flow (EOF) was created. By using a scanning confocal fluorescence microscope and total internal-reflection fluorescence microscope (TIRFM), we demonstrated that negatively charged DNA molecules were focused by the nonuniform EOF into a thin layer at the glass surface. This phenomenon was applied to selectively detect target DNA molecules without requiring the separation of excessive probes and can be applied continuously to achieve high throughput.;A variable-angle-TIRFM was constructed for imaging single DNA molecule dynamics at a solid/liquid interface. Implications we have are that the measured intensities cannot be used directly to determine the distances of molecules from the surface and the experimental counting results depict the distance-dependent dynamics of molecules near the surface. Molecules at low ionic strengths experience electrostatic repulsion at distances much further away from the surface than the calculated thickness of the electrical double layer.;lambda-DNA was employed as a nanoprobe for different functionalized surfaces to elucidate adsorption in chromatography. The 12-base unpaired ends of this DNA provide exposed purine and pyrimidine groups for adsorption. Patterns of self-assembled monolayers (SAMs) and patterns of metal oxides are generated. By recording the real-time dynamic motion of DNA molecules at the SAMs/aqueous interface, the various parameters governing the retention of an analyte during chromatographic separation can be studied. Even subtle differences among adsorptive forces can be revealed.;Dynamic conformational changes of the prosthetic group, flavin adenine dinucleotide (FAD), in flavoprotein NADH peroxidase, in thioredoxin reductase, and in free solution were monitored with TIRFM. FAD bound loosely in the proteins changed from the stacked conformation to the unstacked conformation upon laser excitation. FAD in free solution not only underwent conformational changes but also reacted with each other to form a dimer.;Direct measurement of the single-molecule enzymatic cleavage rates of ApaI-DNA complex in the presence of various concentrations of MgCl2 solution is reported. Results suggest that there exists a distribution of ApaI conformations around the restriction site.</p>
dc.format.mimetype application/pdf
dc.identifier archive/lib.dr.iastate.edu/rtd/1573/
dc.identifier.articleid 2572
dc.identifier.contextkey 6095103
dc.identifier.doi https://doi.org/10.31274/rtd-180813-9895
dc.identifier.s3bucket isulib-bepress-aws-west
dc.identifier.submissionpath rtd/1573
dc.identifier.uri https://dr.lib.iastate.edu/handle/20.500.12876/69391
dc.language.iso en
dc.source.bitstream archive/lib.dr.iastate.edu/rtd/1573/r_3184631.pdf|||Fri Jan 14 20:45:45 UTC 2022
dc.subject.disciplines Analytical Chemistry
dc.subject.disciplines Biochemistry
dc.subject.keywords Chemistry
dc.subject.keywords Analytical chemistry
dc.title Real-time monitoring and manipulation of single bio-molecules in free solution
dc.type article
dc.type.genre dissertation
dspace.entity.type Publication
relation.isOrgUnitOfPublication 42864f6e-7a3d-4be3-8b5a-0ae3c3830a11
thesis.degree.level dissertation
thesis.degree.name Doctor of Philosophy
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