FLUORESCENCE IMAGING DETECTION FOR CAPILLARY ISOELECTRIC-FOCUSING

A simple laser-induced fluorescence (LIF) imaging detector and an ultr asensitive LIF imaging detector are described for capillary isoelectri c focusing (CIEF). An argon ion laser beam of 496.5 nm is used as exci tation source. In the simple LIF imaging detector, the excitation beam is directed into a capillary column by an optic fiber array. In the u ltrasensitive LIF imaging detector, the laser beam is first expanded, then is focused into the 4.5 cm long capillary column by a cylindrical lens. Fluorescence emission is detected by a charge-coupled device (C CD) camera. The feasibility and performance of the LIF imaging detecto r system for CIEF are first verified with a naturally fluorescent prot ein, b-phycoerythrin. Then, the ultrasensitive LIF imaging system is u sed as a detector for CIEF of proteins labeled with fluorescein isothi ocyanate (FITC). Three FITC-labeled proteins (i) alpha-D-galatosylated FITC-albumin, (ii) insulin-FITC, and (iii) casein-FITC, are used as m odel samples. Fluorescence images of the model samples are measured du ring the CIEF process. The focusing of the protein samples is complete in about 1.5 min. The ultrasensitive detector's detection limits for the FITC-labeled proteins are at the level of 10(-10) M, and the mass detection limits are about 4.5 X 10(-17) mole, even though only 10% of the fluorescence emission is collected. Therefore, the method is capa ble of separating and detecting 10(-11) M or amole (10(-18) mole) leve l protein samples with a band-pass filter more specific to the fluores cence light. Potential applications of the LIF imaging system in addit ion to quantitation of separated fluorescent species in various capill ary electrophoresis methods can also include investigation of interact ion between analytes focused in a capillary column.