Continuous-flow single-molecule CE with high detection efficiency

• Published in: Electrophoresis Vol. 28; no. 14; pp. 2430 - 2438
• Main Authors: Schiro, Perry G., Kuyper, Christopher L., Chiu, Daniel T.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01-07-2007; WILEY‐VCH Verlag
• Subjects: Confocal LIF; Electrophoresis, Capillary - methods; Fluorescein-5-isothiocyanate - isolation & purification; Fluorescence correlation spectroscopy; Fluorescent Dyes - isolation & purification; Microchip electrophoresis; Microfluidic Analytical Techniques; Microscopy, Confocal; Sensitivity and Specificity; Single-molecule
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.200600730

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 (∼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 (∼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 μm wide channel we achieved a 94% overall detection efficiency for single Alexa488 dye molecules when a 2 μm×0.5 μm illumination area was used, but only 34% detection efficiency with a 0.5 μm‐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.