Fluorescence imaging detection for capillary isoelectric focusing

Abstract

A simple laser-induced fluorescence (LIF) imaging detector and an ultrasensitive LIF imaging detector are described for capillary isoelectric focusing (CIEF). An argon ion laser beam of 496.5 nm is used as excitation source. In the simple LIF imaging detector, the excitation beam is directed into a capillary column by an optic fiber array. In the ultrasensitive 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 (CCD) camera. The feasibility and performance of the LIF imaging detector system for CIEF are first verified with a naturally fluorescent protein, b-phycoerythrin. Then, the ultrasensitive LIF imaging system is used as a detector for CIEF of proteins labeled with fluorescein isothiocyanate (FITC). Three FITC-labeled proteins (i) α-D-galatosylated FITC-albumin, (ii) insulin-FITC, and (iii) casein-FITC, are used as model samples. Fluorescence images of the model samples are measured during 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 × 10⁻¹⁷ mole, even though only 10% of the fluorescence emission is collected. Therefore, the method is capable of separating and detecting 10⁻¹¹ M or amole (10⁻¹⁸ mole) level protein samples with a band-pass filter more specific to the fluorescence light. Potential applications of the LIF imaging system in addition to quantitation of separated fluorescent species in various capillary electrophoresis methods can also include investigation of interaction between analytes focused in a capillary column.