Miniaturization of ElectrochemicalDetection System for Biuret-Based Peptide Detection Following Capillary HPLC

Miniaturization of liquid chromatography, capillary HPLC, has made possible the separation of samples with volumes in the low microliter to nanoliter range. Downsizing separation column requires more sensitive detection method. Biuret-based electrochemistry has proven to be effective in peptide detection, but work needs to be done to make it feasible in capillary separation system.This thesis presents modifications of a standard electrochemical detector with dual working electrodes, which decreases bandspreading into the nL range suitable for capillary HPLC. This miniaturized detector has detection limit of 3 nM (6 fmol) for peptides on the upstream anode. The downstream cathode has high selectivity by applying reductive potential. The common carbon working electrode can be replaced with gold electrodes with a self-assembled naphthalenethiol monolayer, which suppresses the background current from Au while giving detectable current from Cu-peptide redox reaction. When the modified Au electrode is assembled to a flow cell for peptide detection, it gives stable current response and the sensitivity is comparable to a glassy carbon electrode.These advances will permit non-electrochemists to determine peptides by capillary HPLC quantitatively, and open the way for chip-based detectors