Continuous-flow single-molecule CE with high detection efficiency.
This paper describes the use of two-beam line-confocal detection geometry for measuring the total mobility of individual molecules undergoing continuous-flow CE separation. High-sensitivity single-molecule confocal detection is usually performed with a diffraction limited focal spot (approximately 500 nm in diameter), which necessitates the use of nanometer-sized channels to ensure all molecules flow through the detection volume. To allow for the use of larger channels that are a few micrometers in width, we employed cylindrical optics to define a rectangular illumination area that is diffraction-limited (approximately 500 nm) in width, but a few micrometers in length to match the width of the microchannel. We present detailed studies that compare the performance of this line-confocal detection geometry with the more widely used point-confocal geometry. Overall, we found line-confocal detection to provide the highest combination of signal-to-background ratio and spatial detection efficiency when used with micrometer-sized channels. For example, in a 2 microm wide channel we achieved a 94% overall detection efficiency for single Alexa488 dye molecules when a 2 microm x 0.5 microm illumination area was used, but only 34% detection efficiency with a 0.5 microm-diameter detection spot. To carry out continuous-flow CE, we used two-beam fluorescent cross-correlation spectroscopy where the transit time of each molecule is determined by cross-correlating the fluorescence registered by two spatially offset line-confocal detectors. We successfully separated single molecules of FITC, FITC-tagged glutamate, and FITC-tagged glycine.
Schiro
Perry G
PG
Department of Chemistry, University of Washington, Seattle, WA 98195-1700, USA.
Kuyper
Christopher L
CL
Chiu
Daniel T
DT
D019075:Electrophoresis, Capillary / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000302:isolation & purification; D005456:Fluorescent Dyes / Q000302:isolation & purification; D046210:Microfluidic Analytical Techniques; D018613:Microscopy, Confocal; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D016650:Fluorescein-5-isothiocyanate