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Light‐emitting diode‐induced fluorescence detection of native proteins in capillary electrophoresis
Author(s) -
Sluszny Chanan,
He Yan,
Yeung Edward S.
Publication year - 2005
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200500348
Subject(s) - photomultiplier , capillary electrophoresis , fluorescence , light emitting diode , diode , analytical chemistry (journal) , luminescence , electrophoresis , materials science , detection limit , fluorescence spectroscopy , chemistry , optoelectronics , chromatography , optics , detector , physics
Abstract A continuous‐wave 280 nm light‐emitting diode (LED) was used as the excitation source for native fluorescence detection of proteins in CE. The operating current and temperature of the LED were optimized in order to achieve high luminescence power. It was found that a forward current of 30 mA and a temperature of approximately 5°C gave the best S/N. By using a set of two ball lenses to focus light from the LED, we achieved a spot of approximately 200 μm with a power of 0.1–0.2 mW on the detection window. Fluorescence was collected with a ball lens at 90° angle through a bandpass filter onto a photomultiplier tube. In CZE an LOD of 20 n M for conalbumin was reached. In capillary gel electrophoresis all eight proteins from a commercial standard kit were detected with high S/N. For a 10 μg/mL total protein mixture, S/N was better than 3 for all proteins in solution. Further improvement in LOD should be possible on utilization of an LED with higher luminescence power.