Title

Single molecule fluorescence under conditions of fast flow

RIS ID

104345

Publication Details

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.

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.

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Link to publisher version (DOI)

http://dx.doi.org/10.1021/ac202313d