Abstract
This paper describes a laser‐induced fluorescence (LIF) imaging system for detecting proteins separated by isoelectric focusing (IEF). IEF was carried out in either microfabricated glass devices or in capillaries. Compared to the single‐point‐detection used in capillary IEF (CIEF), the imaging approach eliminates the need for mobilizing the focused proteins to the detector, thereby reducing proteins' de‐focusing during mobilization and improving separation resolution. The LIF imaging system consists of a laser; its diameter is first increased through a beam expander and subsequently the laser beam is focused into a line using a cylindrical lens. The resultant laser line is used to image an entire channel or capillary in which protein separation take place. The fluorescence emission is collected with a cooled, scientific grade charge‐coupled device (CCD) camera. The detection limit was determined using a series of concentrations of fluorescein solutions. The imaging system was demonstrated by isoelectric focusing of proteins, including Green Fluorescent Protein, B‐phycoerythrin, and R‐phycoerythrin. In addition, we can obtain temporal profiles of protein separation, providing useful information about the dynamic behavior of protein migration during IEF. *Portions of this work were presented in “Lab‐on‐a‐chip: platforms, devices, and applications” at the Optics East conference, Philadelphia, PA, Oct., 2004.
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