Continuous‐flow single‐molecule CE with high detection efficiency

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.

Intercellular nanotubes: insights from imaging studies and beyond

“…Once isolated, ICNs could be encapsulated in aqueous droplets and used as nanolabs for the biochemical manipulation 84 and analysis of the composition. 84–86 A schematic work flow involved in using a droplet nanolab approach for the analysis of single cells, organelles, and ICNs is depicted in Figure 8.3.…”

Pressure-driven laminar flow switching for rapid exchange of solution environment around surface adhered biological particles

“…The difference in the switching time of the laminar flow boundary between the flow near the surface and that near the middle of the channel was even greater. To measure more quantitatively the rate at which this front moves across the channel at different depths, we used line-confocal detection 16. Here, the 488 nm laser was focused to a line, and the fluorescence from this line was passed through a slit placed at the image plane and then focused onto a single-photon detector.…”

Capillary and Microchip Electrophoresis

“…Similarly, separations in the millisecond time domain can be followed in the chip format which is operated with gated sample injection [67] and, for a 2 Â 2 mm channel of 5 mm length with LIF detection, microchip CE has been shown to be suitable for single molecule separation and detection of fluorescein isothiocyanate (FITC)-labeled amino acids in the sub-millisecond time domain [182]. Complex mixtures or challenging pairs of analytes require CE instrumentation with extreme resolution.…”