Single-molecule measurements of transient biomolecular complexes through microfluidic dilution

Authors

Mathew H. Horrocks, University of CambridgeFollow
Luke Rajah, University of Cambridge
Peter Jönsson, University of Cambridge
Magnus Kjaergaard, University of Cambridge
Michele Vendruscolo, University of Cambridge
Tuomas P. J Knowles, University of Cambridge
David Klenerman, University of Cambridge

RIS ID

104342

Publication Details

Horrocks, M. H., Rajah, L., Jönsson, P., Kjaergaard, M., Vendruscolo, M., Knowles, T. P. J. & Klenerman, D. (2013). Single-molecule measurements of transient biomolecular complexes through microfluidic dilution. Analytical Chemistry, 85 (14), 6855-6859.

Abstract

Single-molecule confocal microscopy experiments require concentrations which are low enough to guarantee that, on average, less than one single molecule resides in the probe volume at any given time. Such concentrations are, however, significantly lower than the dissociation constants of many biological complexes which can therefore dissociate under single-molecule conditions. To address the challenge of observing weakly bound complexes in single-molecule experiments in solution, we have designed a microfluidic device that rapidly dilutes samples by up to one hundred thousand times, allowing the observation of unstable complexes before they dissociate. The device can interface with standard biochemistry laboratory experiments and generates a spatially uniform dilution that is stable over time allowing the quantification of the relative concentrations of different molecular species.