2002 W.A.E. McBryde Award Lecture — Affinity recognition, capillary electrophoresis, and laser-induced fluorescence polarization for ultrasensitive bioanalysis

Abstract

The combination of affinity recognition, capillary electrophoresis (CE), laser-induced fluorescence (LIF), and fluorescence polarization for the ultrasensitive determination of compounds of biological interest is described. Competitive immunoassays using CE–LIF eliminate the need for fluorescently labeling trace analytes of interest and are particularly useful for determination of small molecules, such as cyclosporine, gentamicin, vancomycin, and digoxin. Fluorescence polarization allows for differentiation of the antibody-bound from the unbound small molecules. Noncompetitive affinity CE–LIF assays are shown to be highly effective in the determination of biomarkers for DNA damage and HIV-1 infection. An antibody (or aptamer) is used as a fluorescent probe to bind with a target DNA adduct (or the reverse transcriptase of the HIV-1 virus), with the fluorescent reaction products being separated by CE and detected by LIF. Aptamers are attractive affinity probes for protein analysis because of high affinity, high specificity, and the potential for a wide range of target proteins. Fluorescence polarization provides unique information for studying molecular interactions. Innovative integrations of these technologies will have broad applications ranging from cancer research, to biomedical diagnosis, to pharmaceutical and environmental analyses.Key words: capillary electrophoresis, laser-induced fluorescence, fluorescence polarization, immunoassay, affinity probes, antibodies, aptamers, DNA damage, toxins, therapeutic drugs.