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Single molecule fluorescence under conditions of fast flow

Journal Article


Abstract


  • We have experimentally determined the optimal flow velocities to characterize or count single molecules by using a simple microfluidic device to perform two-color coincidence detection (TCCD) and single pair Förster resonance energy transfer (spFRET) using confocal fluorescence spectroscopy on molecules traveling at speeds of up to 10 cm s -1. We show that flowing single fluorophores at ≥0.5 cm s -1 reduces the photophysical processes competing with fluorescence, enabling the use of high excitation irradiances to partially compensate for the short residence time within the confocal volume (10-200 μs). Under these conditions, the data acquisition rate can be increased by a maximum of 38-fold using TCCD at 5 cm s -1 or 18-fold using spFRET at 2 cm s -1, when compared with diffusion. While structural characterization requires more photons to be collected per event and so necessitates the use of slower speeds (2 cm s -1 for TCCD and 1 cm s -1 for spFRET), a considerable enhancement in the event rate could still be obtained (33-fold for TCCD and 16-fold for spFRET). Using flow under optimized conditions, analytes could be rapidly quantified over a dynamic range of up to 4 orders of magnitude by direct molecule counting; a 50 fM dual-labeled model sample can be detected with 99.5% statistical confidence in around 8 s using TCCD and a flow velocity of 5 cm s -1. © 2011 American Chemical Society.

UOW Authors


  •   Li, Haitao (external author)
  •   Shim, Jung-uk (external author)
  •   Ranasinghe, Rohan T. (external author)
  •   Huck, Wilhelm T. S. (external author)
  •   Abell, Chris (external author)
  •   Klenerman, David (external author)
  •   Clarke, Richard W. (external author)
  •   Horrocks, Mathew H. (external author)

Publication Date


  • 2012

Citation


  • Horrocks, M. H., Li, H., Shim, J., Ranasinghe, R. T., Clarke, R. W., Huck, W. T. S., Abell, C. & Klenerman, D. (2012). Single molecule fluorescence under conditions of fast flow. Analytical Chemistry, 84 (1), 179-185.

Scopus Eid


  • 2-s2.0-84855391797

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/3360

Has Global Citation Frequency


Number Of Pages


  • 6

Start Page


  • 179

End Page


  • 185

Volume


  • 84

Issue


  • 1

Abstract


  • We have experimentally determined the optimal flow velocities to characterize or count single molecules by using a simple microfluidic device to perform two-color coincidence detection (TCCD) and single pair Förster resonance energy transfer (spFRET) using confocal fluorescence spectroscopy on molecules traveling at speeds of up to 10 cm s -1. We show that flowing single fluorophores at ≥0.5 cm s -1 reduces the photophysical processes competing with fluorescence, enabling the use of high excitation irradiances to partially compensate for the short residence time within the confocal volume (10-200 μs). Under these conditions, the data acquisition rate can be increased by a maximum of 38-fold using TCCD at 5 cm s -1 or 18-fold using spFRET at 2 cm s -1, when compared with diffusion. While structural characterization requires more photons to be collected per event and so necessitates the use of slower speeds (2 cm s -1 for TCCD and 1 cm s -1 for spFRET), a considerable enhancement in the event rate could still be obtained (33-fold for TCCD and 16-fold for spFRET). Using flow under optimized conditions, analytes could be rapidly quantified over a dynamic range of up to 4 orders of magnitude by direct molecule counting; a 50 fM dual-labeled model sample can be detected with 99.5% statistical confidence in around 8 s using TCCD and a flow velocity of 5 cm s -1. © 2011 American Chemical Society.

UOW Authors


  •   Li, Haitao (external author)
  •   Shim, Jung-uk (external author)
  •   Ranasinghe, Rohan T. (external author)
  •   Huck, Wilhelm T. S. (external author)
  •   Abell, Chris (external author)
  •   Klenerman, David (external author)
  •   Clarke, Richard W. (external author)
  •   Horrocks, Mathew H. (external author)

Publication Date


  • 2012

Citation


  • Horrocks, M. H., Li, H., Shim, J., Ranasinghe, R. T., Clarke, R. W., Huck, W. T. S., Abell, C. & Klenerman, D. (2012). Single molecule fluorescence under conditions of fast flow. Analytical Chemistry, 84 (1), 179-185.

Scopus Eid


  • 2-s2.0-84855391797

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/3360

Has Global Citation Frequency


Number Of Pages


  • 6

Start Page


  • 179

End Page


  • 185

Volume


  • 84

Issue


  • 1