Abstract

Two instrumental/assay schemes based on capillary electrophoresis (CE) and laser-induced fluorescence (LIF) detection for sensitive and specific analysis of compounds within biosamples were designed and characterized. Ultraviolet (UV) excitation for LIF detection of underivatized compounds, termed laser-induced-native fluorescence (LINF), represents a simple, sensitive and selective method for detecting CE separated compounds that fluoresce appreciably when excited by UV radiation. A major drawback of CE-LINF systems, however, has been the expense and the complexity of the laser required for excitation in the deep UV wavelength range of 200-300 nm. To this end, the performance of a relatively inexpensive, low-power, pulsed KrF laser operating at 248 nm in a pseudo-continuous wave mode was evaluated as an excitation source for native fluorescence detection of tryptophan. On-column LINF detection limits in the low nanomolar range are obtained for tryptophan with the KrF source, which is similar to that achieved by LINF detectors that use costly large frame frequency-doubled argon ion lasers. The developed CE-LINF instrument is further applied towards the selective profiling of tryptophan-containing proteins and peptides in human serum and saliva, and catecholamines in human urine illustrating the potential use of this system for diagnosing various disease states. The progressive development of a robust immunoassay for the detection of specific antibodies from crude serum is also presented. Off-line immunocapture/immunosubtraction (ICIS) by magnetic particles coupled to subsequent CE-LIF analysis is utilized to detect affinity interactions between solid-phase immobilized host antibodies and selected fluorescent antigens: (i) fluorescein; (ii) fluorescent-labeled neuropeptides and (iii) fluorescent-labeled peptides representing antigenic p24 sequences.