Continuous-flow single-molecule CE with high detection efficiency

Electrophoresis. 2007 Jul;28(14):2430-8. doi: 10.1002/elps.200600730.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Electrophoresis, Capillary / methods*
  • Fluorescein-5-isothiocyanate / isolation & purification
  • Fluorescent Dyes / isolation & purification
  • Microfluidic Analytical Techniques*
  • Microscopy, Confocal*
  • Sensitivity and Specificity

Substances

  • Fluorescent Dyes
  • Fluorescein-5-isothiocyanate