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Fiber-optic ultraviolet resonance raman spectroscopy for biophysical investigations Barbosa, Christopher James
Abstract
The study of molecules with biological significance has benefited from the development of numerous analytical techniques. However, this field of study is broad and complex such that it is generally not adequately served by any one technique alone. This thesis describes the further development and application of fiber-optic coupled ultraviolet resonance Raman spectroscopy (FO-UVRRS) as an additional bioanalytical and biophysical tool. FO-UVRRS has been hampered by both fundamental problems inherent to the resonance Raman effect and by practical constraints that can generally be attributed to the nature of the more common U V light sources. Optimization of the instrument, including upgraded U V laser technology coupled with improved optical fiber materials and refinement of the fiber optic probe, designed specifically for UVRRS and unique to this laboratory, has resulted in dramatic advances in spectrometer performance over the course of this research. Computer simulations have been used to improve fiber optic probe performance by 50% and quantify a power distribution advantage over conventional sampling apparatus for UVRRS. It is shown that the fiber optic probe design used here allows relatively high power to be delivered to the sample without causing photodamage. This feature of FO-UVRRS is exploited to accurately record the Raman scattering crosssections of 2'-deoxyribonucleosides under conditions which are shown to be free of photo-induced damage or Raman saturation effects. The advances reported here have expanded the applicability of UVRRS. It is demonstrated that FO-UVRRS is capable of probing bound aromatic substrates in a light sensitive enzyme system under strictly anaerobic conditions if necessary. It is definitively shown that 2,3-dihydroxybiphenyl 1,2-dioxygenase (EC 1.13.11.39) binds its catecholic substrate, 2,3-dihydroxybiphenl, as a monoanion, which is a significant result because of the inability to confirm this aspect of the enzyme mechanism by any other analytical method to date. Furthermore, in support of the spectral interpretation of 2,3- dihydroxybiphenl, the spectra of catechol itself have been recorded in neutral, monoanionic and dianionic forms free in solution. Together, this work demonstrates not only new applications of FO-UVRRS but also the superiority of the specially designed fiber-optic probes as the sampling tool used for these investigations.
Item Metadata
Title |
Fiber-optic ultraviolet resonance raman spectroscopy for biophysical investigations
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
2002
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Description |
The study of molecules with biological significance has benefited from the development
of numerous analytical techniques. However, this field of study is broad and complex
such that it is generally not adequately served by any one technique alone. This thesis
describes the further development and application of fiber-optic coupled ultraviolet
resonance Raman spectroscopy (FO-UVRRS) as an additional bioanalytical and
biophysical tool.
FO-UVRRS has been hampered by both fundamental problems inherent to the resonance
Raman effect and by practical constraints that can generally be attributed to the nature of
the more common U V light sources. Optimization of the instrument, including upgraded
U V laser technology coupled with improved optical fiber materials and refinement of the
fiber optic probe, designed specifically for UVRRS and unique to this laboratory, has
resulted in dramatic advances in spectrometer performance over the course of this
research. Computer simulations have been used to improve fiber optic probe
performance by 50% and quantify a power distribution advantage over conventional
sampling apparatus for UVRRS. It is shown that the fiber optic probe design used here
allows relatively high power to be delivered to the sample without causing photodamage.
This feature of FO-UVRRS is exploited to accurately record the Raman scattering crosssections
of 2'-deoxyribonucleosides under conditions which are shown to be free of
photo-induced damage or Raman saturation effects.
The advances reported here have expanded the applicability of UVRRS. It is
demonstrated that FO-UVRRS is capable of probing bound aromatic substrates in a light
sensitive enzyme system under strictly anaerobic conditions if necessary. It is
definitively shown that 2,3-dihydroxybiphenyl 1,2-dioxygenase (EC 1.13.11.39) binds its
catecholic substrate, 2,3-dihydroxybiphenl, as a monoanion, which is a significant result
because of the inability to confirm this aspect of the enzyme mechanism by any other
analytical method to date. Furthermore, in support of the spectral interpretation of 2,3-
dihydroxybiphenl, the spectra of catechol itself have been recorded in neutral,
monoanionic and dianionic forms free in solution. Together, this work demonstrates not
only new applications of FO-UVRRS but also the superiority of the specially designed
fiber-optic probes as the sampling tool used for these investigations.
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Extent |
10553930 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-09-29
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0061349
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
2002-11
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Item Media
Item Citations and Data
Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.