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

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

^*Corresponding author for this work

School of Engineering

Research output: Chapter in Book/Report/Conference proceeding › Conference contribution

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 microscopy (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.

Original language	English
Title of host publication	Proceedings Volume 7593, Microfluidics, BioMEMS, and Medical Microsystems VIII
Publisher	SPIE
ISBN (Print)	9780819479891
DOIs	https://doi.org/10.1117/12.840035
Publication status	Published - 26 Feb 2010
Event	Microfluidics, BioMEMS, and Medical Microsystems VIII - San Francisco, CA, United States Duration: 25 Jan 2010 → 27 Jan 2010

Publication series

Name	Progress in Biomedical Optics and Imaging - Proceedings of SPIE
Volume	7593
ISSN (Print)	1605-7422

Conference

Conference	Microfluidics, BioMEMS, and Medical Microsystems VIII
Country/Territory	United States
City	San Francisco, CA
Period	25/01/10 → 27/01/10

Keywords / Materials (for Non-textual outputs)

Anisotropy imaging
CFD
FLIM
Microfluidics

Access to Document

10.1117/12.840035

Cite this

Robinson, T., Manning, H. B., Dunsby, C., Neil, M. A. A., Baldwin, G. S., De Mello, A. J., & French, P. M. W. (2010). Investigating fast enzyme-DNA kinetics using multidimensional fluorescence imaging and microfluidics. In Proceedings Volume 7593, Microfluidics, BioMEMS, and Medical Microsystems VIII Article 759304 (Progress in Biomedical Optics and Imaging - Proceedings of SPIE; Vol. 7593). SPIE. https://doi.org/10.1117/12.840035

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title = "Investigating fast enzyme-DNA kinetics using multidimensional fluorescence imaging and microfluidics",

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 microscopy (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.",

keywords = "Anisotropy imaging, CFD, FLIM, Microfluidics",

author = "Tom Robinson and Manning, {Hugh B.} and Christopher Dunsby and Neil, {Mark A.A.} and Baldwin, {Geoff S.} and {De Mello}, {Andrew J.} and French, {Paul M.W.}",

year = "2010",

month = feb,

day = "26",

doi = "10.1117/12.840035",

language = "English",

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series = "Progress in Biomedical Optics and Imaging - Proceedings of SPIE",

publisher = "SPIE",

booktitle = "Proceedings Volume 7593, Microfluidics, BioMEMS, and Medical Microsystems VIII",

address = "United States",

note = "Microfluidics, BioMEMS, and Medical Microsystems VIII ; Conference date: 25-01-2010 Through 27-01-2010",

}

Robinson, T, Manning, HB, Dunsby, C, Neil, MAA, Baldwin, GS, De Mello, AJ & French, PMW 2010, Investigating fast enzyme-DNA kinetics using multidimensional fluorescence imaging and microfluidics. in Proceedings Volume 7593, Microfluidics, BioMEMS, and Medical Microsystems VIII., 759304, Progress in Biomedical Optics and Imaging - Proceedings of SPIE, vol. 7593, SPIE, Microfluidics, BioMEMS, and Medical Microsystems VIII, San Francisco, CA, United States, 25/01/10. https://doi.org/10.1117/12.840035

Investigating fast enzyme-DNA kinetics using multidimensional fluorescence imaging and microfluidics. / Robinson, Tom; Manning, Hugh B.; Dunsby, Christopher et al.
Proceedings Volume 7593, Microfluidics, BioMEMS, and Medical Microsystems VIII. SPIE, 2010. 759304 (Progress in Biomedical Optics and Imaging - Proceedings of SPIE; Vol. 7593).

Research output: Chapter in Book/Report/Conference proceeding › Conference contribution

TY - GEN

T1 - Investigating fast enzyme-DNA kinetics using multidimensional fluorescence imaging and microfluidics

AU - Robinson, Tom

AU - Manning, Hugh B.

AU - Dunsby, Christopher

AU - Neil, Mark A.A.

AU - Baldwin, Geoff S.

AU - De Mello, Andrew J.

AU - French, Paul M.W.

PY - 2010/2/26

Y1 - 2010/2/26

N2 - 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 microscopy (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.

AB - 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 microscopy (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.

KW - Anisotropy imaging

KW - CFD

KW - FLIM

KW - Microfluidics

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DO - 10.1117/12.840035

M3 - Conference contribution

AN - SCOPUS:79958150267

SN - 9780819479891

T3 - Progress in Biomedical Optics and Imaging - Proceedings of SPIE

BT - Proceedings Volume 7593, Microfluidics, BioMEMS, and Medical Microsystems VIII

PB - SPIE

T2 - Microfluidics, BioMEMS, and Medical Microsystems VIII

Y2 - 25 January 2010 through 27 January 2010

ER -

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

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