Title page for ETD etd-07022007-115206


Type of Document Dissertation
Author Hooper, Stephanie Elaine
Author's Email Address shooper@vt.edu
URN etd-07022007-115206
Title Development of an Ionically-Assembled On-Column Enzyme Reactor for Capillary Electrophoresis
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
Anderson, Mark R. Committee Chair
Brewer, Karen J. Committee Member
Long, Gary L. Committee Member
Morris, John R. Committee Member
Yee, Gordon T. Committee Member
Keywords
  • electrochemical detection
  • glutamate oxidase
  • capillary electrophoresis
  • glucose oxidase
  • enzyme reactor
Date of Defense 2007-06-26
Availability unrestricted
Abstract

This work describes the integration of a separation capillary for capillary electrophoresis (CE) with an on-column enzyme reactor for selective determination of the enzyme substrate. The enzyme reaction occurs during a capillary separation, allowing selective determination of the substrate in complex samples without the need for pre- or post- separation chemical modification of the analyte. The overall goal of this work is to develop a system in which sample introduction, separation of the analyte/substrate from other biological species, enzymatic conversion of the analyte/substrate into a detectable product, and sensitive detection are all included within a single analysis scheme.

Immobilization of the enzyme is achieved by electrostatic assembly of poly(diallydimethylammonium chloride) (PDDA) followed by adsorption of a mixture of the negatively charged enzyme glucose oxidase (GOx) and anionic poly(styrenesulfonate) (PSS). The reaction of glucose with the immobilized glucose oxidase produces H2O2 which migrates the length of the capillary under the influence of electroosmotic flow and is detected amperometrically at the capillary outlet.

The optimal response, kinetics, and stability for the enzyme reactor are determined through characterization of several parameters including the concentration ratio of PSS:GOx, applied separation voltage, and the inner diameter of the separation capillary. Various analyte mixtures containing the substrate and other biological species were evaluated to illustrate selective separation and determination of the substrate from other biomolecules. Optimization of this electrostatically assembled capillary enzyme reactor lead to application of these parameters to similar enzymes such as glutamate oxidase. Future application to similar enzymes like L-amino acid oxidase and possible microfluidic systems is a long-term goal of the system.

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