Identifier

etd-07022008-105747

Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

This work describes the application of PMMA-based microanalytical devices for the affinity-type preconcentration of posttranslational modified proteins (PTMs). The choice of poly(methyl methacrylate), PMMA, is based on its biocompatibility, its functional methyl ester group for potential modification, and its extensive applications to create biological microelectromechanical systems (BioMEMS). Developing methodologies for preconcentration of PTMs is important for cancer diagnosis due to PTMs’ influence in the regulatory mechanism underlying the early stage of apoptosis or regulated cell death. Towards this goal, nitroavidin which can reversibly binds to biotin (and biotinylated proteins), was prepared using reported procedure and was characterized using several techniques such as UV-Visible spectroscopy, sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), and Western blot experiments. UV-Visible spectroscopy experiments showed reversible binding of nitroavidin towards the biotin analogue 2-(4’-hydroxyazobenzene) benzoic acid, HABA. From mass spectrometry studies, nitrotyrosine was confirmed to be present in the prepared nitroavidin through an observed photoinduced chemical fragmentation. SPR experiments revealed decrease in binding of nitroavidin towards biotinylated proteins (the equilibrium dissociation constant obtained for the biotin−nitroavidin interactions is higher, KD = 4 x 10–6 M, than biotin-avidin interactions, KD = 1 x 10–13 M). Also, there was an observed efficiency of 23 ± 1% for the capture process of biotinylated proteins on nitroavidin−functionalized PMMA open microchannels, while high capture efficiency (96 ± 0.5%) for bound biotinylated proteins were observed on PMMA microchannels with fabricated microposts. To further improve the efficiency of capture and release processes, PMMA ultra-high-aspect-ratio nanostructures (UHRANs) were employed to provide higher surface-to-volume reactor bed. These PMMA UHRANs were fabricated in our group using previously reported template-based anodization. PMMA nanopillars or nanoposts were developed using photopolymerization between the methyl methacrylate monomer and initiator, while PMMA nanotubes were fabricated using PMMA melt. These nanostructures were UV-modified to promote formation of surface carboxylic acids (pendant −COOH). The confirmation of surface –COOH functionalization on these surfaces was achieved using different surface labeling techniques such as thallium (I) ethoxide and sulfosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate (sulfo-SDTB) and were determined using several techniques such as confocal fluorescence microscopy, UV-Visible spectroscopy, AFM, SEM, and XPS.

Date

2008

Document Availability at the Time of Submission

Secure the entire work for patent and/or proprietary purposes for a period of one year. Student has submitted appropriate documentation which states: During this period the copyright owner also agrees not to exercise her/his ownership rights, including public use in works, without prior authorization from LSU. At the end of the one year period, either we or LSU may request an automatic extension for one additional year. At the end of the one year secure period (or its extension, if such is requested), the work will be released for access worldwide.

Committee Chair

Robin L. McCarley

Included in

Chemistry Commons

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