Electrophoretic separation and confocal laser-induced fluorescence detection at ultralow concentrations in constricted fused-silica capillaries.
Laser-induced fluorescence detection of labeled amino acids in a flowing stream at femtomolar (10(-15)M) concentrations was achieved by using a fused-silica capillary flow-cell comprising a constricted thin-walled detection region with inner diameters (IDs) ranging from 2 to 8 microm. The diameter of the constricted region was made to match a diffraction-limited focus of a uniphase transverse electromagnetic mode (TEM(00)) laser beam. Optimization of capillary dimensions and geometries (i.e., curvature, wall thickness, and outer-inner diameter ratio were performed in order to minimize cylindrical lensing of the focused laser beam. The fluorescence was collected in a confocal optical setup using a 1.3 numerical aperture (NA), 100x oil-immersion objective and a single-photon-counting avalanche diode (SPAD). Under conditions of fluid flow, the constriction in the capillary forces all analytes to traverse across the laser probe volume, resulting in a high sampling efficiency. Fluorescein isothiocyanate-labeled glutamate (FITC-Glu) was electrophoretically separated and detected in capillaries having an ID of 2 microm at the constricted region with detection limits of 250 fM (signal-to-noise ratio (S/N) = 3) in the injected solution.
Lundqvist
Anders
A
Department of Physical Chemistry and Microtechnology Center, Chalmers University of Technology, Göteborg, Sweden.
Chiu
Daniel T
DT
Orwar
Owe
O
D000596:Amino Acids / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D005453:Fluorescence; D005456:Fluorescent Dyes; D007834:Lasers; D018613:Microscopy, Confocal; D012822:Silicon Dioxide
D000596:Amino Acids; D005456:Fluorescent Dyes; D012822:Silicon Dioxide