Highly Efficient Single Molecule Detection in Different Micro and submicrometer Channels with cw-excitation

Abstract

In this article we describe a detailed comparison between single molecule detection in three different types of microchannels. The overall detection efficiencies and the signal-to-noise ratios are compared. A new data evaluation technique that is used is explained in detail. The average number of photons per molecule is calculated from the measurements and the detection properties of each channel with respect to the diffusional characteristics of the dye molecules are investigated. From the detection efficiencies advantages and drawbacks of the individual methods are elucidated as well as possible applications of the techniques are illustrated.

Ultrasensitive on-column laser-induced fluorescence in capillary electrophoresis using multiparameter confocal detection

“…Such designs force all analytes to flow through a tightly focused excitation beam, thus improving the MCE. Examples of micro-channel designs include custom-made pinched capillaries, 17 microfluidic chips, [18][19][20][21] etched glass channels, 22 patch-clamp pipettes, 23,24 and quartz capillaries. 23 The use of micro-channels often hinders diffusion in 1 or 2 dimensions, thus beneficially causing molecules to reside in the detection volume longer and emit more photons.…”

“…Examples of micro-channel designs include custom-made pinched capillaries, 17 microfluidic chips, [18][19][20][21] etched glass channels, 22 patch-clamp pipettes, 23,24 and quartz capillaries. 23 The use of micro-channels often hinders diffusion in 1 or 2 dimensions, thus beneficially causing molecules to reside in the detection volume longer and emit more photons. 23,24 Single molecule fluorescence bursts with S/N z 50 were recorded; 18 however, the drawback of the method is that small-size channels are more prone to contamination and clogging.…”

“…23 The use of micro-channels often hinders diffusion in 1 or 2 dimensions, thus beneficially causing molecules to reside in the detection volume longer and emit more photons. 23,24 Single molecule fluorescence bursts with S/N z 50 were recorded; 18 however, the drawback of the method is that small-size channels are more prone to contamination and clogging. 19,25,26 Consequently, there is only one micro-channel study involving a complex biological sample (cell lysate) which avoids the aforementioned troubles by using a relatively wide channel (1.8 mm  60 mm).…”

“…19 In addition, the shape and material of micro-channels often differ from that of standard fused silica capillaries. This difference may introduce changes to analyte migration speeds and retention times, 17,23 analyte velocity profiles, 19,27 and electro-osmotic flows from those determined in a standard round capillary. The variations in these parameters can make transferring protocols from commercial instruments to micro-channel devices non-trivial.…”

Focal Volume Confinement by Submicrometer-Sized Fluidic Channels

“…The use of microfabricated channels for single-fluorophore detection with high-throughput has been demonstrated previously, and various channel or capillary geometries with dimensions of one to several micrometers diameter have been evaluated. ,− The width of the focal volume was either expanded to span the entire channel width, or only a fraction of the channel was illuminated, and thus, not all molecules flowing through the channel were detected. The upper limit in concentration to avoid simultaneous passage of two or more molecules through the observation volume was therefore similar or worse compared to diffraction-limited confocal optics.…”