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(-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.

Fundamentals and practice for ultrasensitive laser‐induced fluorescence detection in microanalytical systems

“…Imaging the same fluid volume through a much thicker glass volume, as in a 50 mm id/360 mm od capillary, produced much worse performance. One problem with such small dimensions is that, at ordinary flowrates, the probe volume can be too short, resulting in residence times that are nonoptimal [66]. For a 1 mm probe volume diameter, a molecule traveling at an ordinary electrophoresis speed of 1 mm/s will transit the probe volume in 1 ms.…”

“…In this case, the capillary was pulled to 0.5-0.6 mm, so the 1-2 fL probe volume matched the capillary well. Lundqvist, Chiu, and Orwar [66] also used an oil immersion objective and small aperture with pulled capillaries to confine the sample stream to the 14 fL probe volume. Optical calculations were used to optimally match their capillary dimensions (8 mm id/45 mm od) to the probe volume and to minimize imaging aberrations.…”

Direct fluorometric detection of paramagnetic and heavy metal ions at sub‐amol level using an aromatic polyaminocarboxylate by CZE: Combination of pre‐ and on‐capillary complexation technique

“…The derivatization of amino function using FITC-I is well known, in particular, for detecting protein and amino acids [43][44][45][46]. While the reaction proceeded easily, the purity of the product was very sensitive in terms of the pH value, temperature, and reaction time.…”

Chemical cytometry on microfluidic chips

“…Several groups have attempted to solve this problem by decreasing the dimensions of the channel to the nanometer range so that the entire cross-section fits into the focus of the confocal microscope [95,96]. Zare et al [8] widened the excitation laser focus in one direction by using a cylindrical optics (as shown in Fig.…”