Investigating fast enzyme-DNA kinetics using multidimensional fluorescence imaging and microfluidics

Tom Robinson; Hugh B. Manning; Christopher Dunsby; Mark A. A. Neil; Geoff S. Baldwin; Andrew J. de Mello; Paul M. W. French

doi:10.1117/12.840035

26 February 2010 Investigating fast enzyme-DNA kinetics using multidimensional fluorescence imaging and microfluidics

Tom Robinson, Hugh B. Manning, Christopher Dunsby, Mark A. A. Neil, Geoff S. Baldwin, Andrew J. de Mello, Paul M. W. French

Author Affiliations +

Proceedings Volume 7593, Microfluidics, BioMEMS, and Medical Microsystems VIII; 759304 (2010) https://doi.org/10.1117/12.840035
Event: SPIE MOEMS-MEMS, 2010, San Francisco, California, United States

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.

Citation Download Citation

Tom Robinson, Hugh B. Manning, Christopher Dunsby, Mark A. A. Neil, Geoff S. Baldwin, Andrew J. de Mello, and Paul M. W. French "Investigating fast enzyme-DNA kinetics using multidimensional fluorescence imaging and microfluidics", Proc. SPIE 7593, Microfluidics, BioMEMS, and Medical Microsystems VIII, 759304 (26 February 2010); https://doi.org/10.1117/12.840035

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