Electrophoretic separation and confocal laser‐induced fluorescence detection at ultralow concentrations in constricted fused‐silica capillaries

Abstract
Laser‐induced fluorescence detection of labeled amino acids in a flowing stream at femtomolar (10−15M) 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 μm. The diameter of the constricted region was made to match a diffraction‐limited focus of a uniphase transverse electromagnetic mode (TEM00) 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), 100× 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 μm at the constricted region with detection limits of 250 fM (signal‐to‐noise ratio (S/N) = 3) in the injected solution.