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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.

The main goal of this research was to develop an easy to prepare and sensitive biosensor that would be able to detect glutamate in solution using ionic self-assembly methods. This was accomplished by preparing an ionically-self-assembled monolayer that included an electron transport mediator and an enzyme that would generate a current proportional to the concentration of analytes in solution. Biosensors were produced for the detection of glucose and glutamate. Ferrocene poly(allylamine) (FePAA) was assembled on negatively charged self-assembled monolayer and shown to be electrostatically bound by cyclic voltammetry. Model films were made of FePAA and poly(styrenesulfonate) to determine if multilayer films could be assembled using electrostatic assembly. These experiments demonstrated that 7 bilayers is the maximum number of bilayers oxidizable by the heterogeneous reaction at the electrode surface. ISAMs were then assembled on a 2 mm gold electrode and on a gold fiber microelectrode using FePAA and glucose oxidase. Using cyclic voltammetry, these ISAMs were shown to be able to oxidize glucose in solution. The LOD was determined to be lower for the microelectrode than for the 2 mm gold electrode, which was expected, while both compared well to the literature. The Km? were found to be smaller than other glucose biosensors while the Icat increased with increasing number of bilayers. This demonstrated that the GluOx is making good electrical contact with the layer below. These glucose oxidase ISAMs, however, do not exhibit structural stability in flow-injection experiments. As a solution to the ISAM modified electrodes degrading in the flowing system, a covalently modified surface was developed. Using cyclic voltammetry, these covalently modified surfaces were shown to be able to oxidize glucose in solution. The LOD of the covalently modified 2 mm gold electrode was calculated to be lower than the 2 mm ISAM modified gold electrode, due to the fast heterogeneous kinetics on the covalently modified electrode surface. The Km? and Icat for the covalently modified 2 mm gold electrode were found to be the similar to the 2 mm ISAM modified gold electrode indicating that the covalently modified electrodes will be a suitable replacement. The covalently modified surfaces exhibit excellent structural stability and detect much lower glucose amounts in flow-injection experiments. ISAMs were subsequently assembled on gold fiber microelectrodes using FePAA and glutamate oxidase. Glutamate was able to be detected in solution at biologically significantly quantities using cyclic voltammetry. The Km? was shown to be comparable to literature values and Icat was shown to increase with increasing number of bilayers. These results demonstrate that an ISAM constructed using FePAA/GlutOx is a feasible way to detect glutamate in a system.