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Proceedings Paper

Investigating fast enzyme-DNA kinetics using multidimensional fluorescence imaging and microfluidics
Author(s): Tom Robinson; Hugh B. Manning; Christopher Dunsby; Mark A. A. Neil; Geoff S. Baldwin; Andrew J. de Mello; Paul M. W. French
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Paper Abstract

We have developed a rapid microfluidic mixing device to image fast kinetics. To verify the performance of the device it was simulated using computational fluid dynamics (CFD) and the results were directly compared to experimental fluorescence lifetime imaging (FLIM) measurements. The theoretical and measured mixing times of the device were found to be in agreement over a range of flow rates. This mixing device is being developed with the aim of analysing fast enzyme kinetics in the sub-millisecond time domain, which cannot be achieved with conventional macro-stopped flow devices. Here we have studied the binding of a DNA repair enzyme, uracil DNA glycosylase (UDG), to a fluorescently labelled DNA substrate. Bulk phase fluorescence measurements have been used to measure changes on binding: it was found that the fluorescence lifetime increased along with an increase in the polarisation anisotropy and rotational correlation time. Analysis of the same reaction in the microfluidic mixer by CFD enabled us to predict the mixing time of the device to be 46 μs, more than 20 times faster than current stopped-flow techniques. We also demonstrate that it is possible to image UDG-DNA interactions within the micromixer using the signal changes observed from the multidimensional spectrofluorometer.

Paper Details

Date Published: 26 February 2010
PDF: 8 pages
Proc. SPIE 7593, Microfluidics, BioMEMS, and Medical Microsystems VIII, 759304 (26 February 2010); doi: 10.1117/12.840035
Show Author Affiliations
Tom Robinson, Imperial College London (United Kingdom)
Hugh B. Manning, Imperial College London (United Kingdom)
Christopher Dunsby, Imperial College London (United Kingdom)
Mark A. A. Neil, Imperial College London (United Kingdom)
Geoff S. Baldwin, Imperial College London (United Kingdom)
Andrew J. de Mello, Imperial College London (United Kingdom)
Paul M. W. French, Imperial College London (United Kingdom)

Published in SPIE Proceedings Vol. 7593:
Microfluidics, BioMEMS, and Medical Microsystems VIII
Holger Becker; Wanjun Wang, Editor(s)

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