Development of methodology and miniaturized instrumentation for peptide analysis

The ability to study metabolism, transport and release of peptides at biologically relevant concentrations is important for the design of more effective pharmaceuticals. The research described in this thesis summary focuses on the development of sensitive and selective methodology for the determination of peptides in biological matrices. This method exploits the ability of copper to form electroactive complexes with peptides. The optimal reaction and separation conditions for angiotensin and its metabolites have been determined using capillary electrophoresis with UV detection. CE combined with electrochemical detection (EC) provides a sensitive and selective technique for the detection of many biologically important compounds including peptides. The use of copper complexation in conjunction with CE-EC has the advantage over conventional derivatization based methods for peptides since it does not require the presence of a free amine group.; After the initial studies, work focused on the development of microchip CE devices for the detection of peptides. Significant work has been done on the development of poly(dimethylsiloxane) (PDMS) as a microchip substrate; as opposed to glass which is typically reported for microstructure fabrication. A comparative study of a glass versus a PDMS microchip substrate for the analysis of FITC-labeled peptides and amino acids has been obtained using laser-induced fluorescence (LIF) detection. Separations with lower efficiencies and increased LODs for hydrophobic peptides were obtained using polymer substrates. To circumvent this adsorption problem, methods to modify the surface of the polymer were undertaken. In nearly all microfluidic CE with amperometric detection applications, electrode alignment usually occurs in an end-channel configuration, leading to peak dispersion. In order to prevent losses in efficiency witnessed by this type of alignment, off channel detection was performed using a Pd decoupler. With this technique, the separation field is grounded prior to reaching the working electrode, allowing the working electrode to be placed in the separation channel, effectively limiting band broadening. Eventually, these micro-systems will be used in conjunction with cell culture or microdialysis sampling for the investigation of peptide metabolism and transport.