James C. Sternberg

Semiconductor laser-induced fluorescence detection in capillary electrophoresis using a cyanine dye

Cy5, an activated carboxyl cyanine fluorophore, was characterized by capillary electrophoresis (CE) using a semiconductor laser at 652 nm to induce fluorescence. Hydrolysis of the activated Cy5 in the presence of ammonia results in the formation of a mono- and diamide and a dicarboxylic acid. A Cy5-labeled oligonucleotide M13 primer for DNA sequencing (M13mp18 template) was synthesized with a purity of better than 95%. The labeled primer was analyzed by liquid chromatography, using UV-visible detection, and by CE, monitored by laser-induced fluorescence (LIF) detection. Analysis of the Cy5-labeled oligonucleotide primer by CE-LIF in a 9% polyacrylamide gel-filled capillary indicated the purity of the major Cy5-oligonucleotide primer was greater than 90%. The detection sensitivity for Cy5-based CE-LIF detection system with a 2.5-mW red semiconductor laser is about 10(-10) M.

Nephelometric Inhibition Immunoassay for Small Molecules

The use of nephelometry, and especially rate nephelometry (Tiffany, Parella, Johnson and Burtis, 1974), for the measurement of specific serum proteins has become very well established. In nephelometric methods, the marked increases in light scattering which occur as a consequence of the immunochemical reaction between polyvalent antigens (such as proteins) and their antibodies is utilized. Thus, while solutions of protein molecules scatter only a small to moderate amount of visible light, their reactions with their specific antisera lead to the formation of crosslinked three dimensional immuno complexes large enough to scatter a significantly increased amount of light, and, eventually, to precipitate. In rate nephelometry, the rate of increase of light scattering during the course of this reaction is utilized to provide a measure of the protein concentration.