Anal. Chem. 2007, 79, 4101-4109
Quantitative Kinetic Analysis in a Microfluidic
Device Using Frequency-Domain Fluorescence
Lifetime Imaging
Sine´ ad M. Matthews,† Alan D. Elder,† Kamran Yunus,† Clemens F. Kaminski,† Colin M. Brennan,‡ and
Adrian C. Fisher*,†
Department of Chemical Engineering, University of Cambridge, New Museums Site, Pembroke Street,
Cambridge, CB2 3RA, UK, and Huddersfield Manufacturing Centre, Syngenta Ltd., P.O. Box A38, Leeds Road,
Huddersfield, HD2 1FF, UK
In this article, we describe the application of fluorescence
lifetime imaging for the quantification of reaction processes
occurring within a microfluidic device. The lifetime is sensitive
to a molecule’s interactions with its surroundings and can be used
to provide quantitative information on the local environment such
as pH changes,10 diffusional mobility,11 conformational changes,12
and quenching.13 This sensitivity enables species that cannot be
resolved using spectral techniques to be discriminated using their
lifetimes.14 Within a microfluidic device, it is relatively simple to
control conditions such as the pH,15 ion concentration,16 and
temperature,17 thus making FLIM a powerful analytical technique.
Previous studies have demonstrated the use of FLIM for the
quantification of processes such as molecular mixing and diffusion
within microchannels.9,18 Recently, we reported the quantitative
analysis of the quenching of Rhodamine 6G by potassium iodide
using FLIM.19,20
In order to extract quantitative mechanistic and kinetic
parameters from spectroscopic data, it is necessary to simulate
the combined mass transport and reaction kinetic processes within
a specific device. Recently we have demonstrated the quantifica-
tion of combined fluid dynamic and mass transport problems
within microfluidic devices where electrochemical analysis is
performed.21,22 While direct analytical solution of the governing
A novel microfluidic approach for the quantification of
reaction kinetics is presented. A three-dimensional finite
difference numerical simulation was developed in order
to extract quantitative kinetic information from fluores-
cence lifetime imaging experimental data. This approach
was first utilized for the study of a fluorescence quenching
reaction within a microchannel; the lifetime of a fluoro-
phore was used to map the diffusion of a quencher across
the microchannel. The approach was then applied to a
more complex chemical reaction between a fluorescent
amine and an acid chloride, via numerical simulation the
bimolecular rate constant for this reaction was obtained.
The potential advantages of using a microfluidic approach for
the study of a number of physical processes such as diffusion,1
phase transfer,2 protein folding,3 and reaction kinetics4 have been
well documented. The high transport rates within microchannels
enables the study of rapid kinetic processes within liquid media.
A range of different in situ analytical techniques have been
developed for the quantification of kinetic and fluid dynamic
processes within microfluidic devices including; confocal fluores-
cence microscopy,5 magnetic resonance imaging,6 optical coher-
ence tomography,7 fluorescence correlation spectroscopy,8 and
fluorescent lifetime imaging microscopy (FLIM).9
Tel.: +44 (0) 1223 763996. Fax: +44 (0) 1223 767407.
† University of Cambridge.
(10) Lin, H. J.; Herman, P.; Lakowicz, J. R. Cytometry, Part A 2003, 52 (2), 77-
89.
(11) Clayton, A. H.; Hanley, Q. S.; Arndt-Jovin, D. J. Biophys. J. 2002, 83 (3),
1631-1649.
‡ Syngenta Ltd.
(1) Kamholz, A. E.; Schilling, E. A.; Yager, P. Biophys. J. 2001, 80, 1967-1972.
(2) Kim, H. B.; Ueno, K.; Chiba, M.; Kogi, O.; Kitamura, N. Anal. Sci. 2000,
16, 871-876.
(12) Calleja, V.; Ameer-Beg, S. M.; Vojnovic, B. Biochem. J. 2003, 372, 33-40.
(13) Szmacinski, H.; Lakowicz, J. R. Sens. Actuator, B: Chem. 1995, 29 (1-3),
16-24.
(3) Bilsel, O.; Kayatekin, C.; Wallace, L. A.; Matthews, C. R. Rev. Sci. Instrum.
2005, 76, 014302.
(4) Cao, C.; Xia, G.; Holladay, J.; Jones, E.; Wang, Y. Appl. Catal., A 2004,
262, 19-29.
(5) Ismagilov, R. F.; Stroock, A. D.; Kenis, P. J. A.; Whitesides, G.; Stone. H. A.
Appl. Phys. Lett. 2000, 76, 2376-2378.
(14) Dowling, K.; Dayel, M. J.; Lever, M. J.; French, P. M. W.; Hares, J. D.;
Dymoke-Bradshaw, A. K. L. Opt. Lett. 1998, 23, 810-812.
(15) Cabrera, C. R.; Finlayson, B.; Yager, P. Anal. Chem. 2001, 73, 658-666.
(16) Yang, M.; Yang, J.; Li, C. W.; Zhao, J. Lab Chip 2002, 2, 158-163.
(17) Mao, H.; Yang, T.; Cremer, P. S. J. Am. Chem. Soc 2002, 124(16), 4432-
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(6) Wensink, H.; Bebito-Lopez, F.; Hermes, D. C.; Verboom, W.; Gardeniers,
H. J. G. E.; Reinhoudt, D. N.; van den Berg, A. Lab Chip 2005, 5, 281-284.
(7) Ahn, Y. C.; Jung, W.; Zhang, J.; Chen. Z. Opt. Express 2005, 13, 8164-
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(8) Kuricheti, K. K.; Buschnmann, V.; Brister, P.; Weston. K. D. In Microfluidics,
BioMEMS, and Medical Microsystems II; Woias, P., Papautsky, I., Eds.; SPIE
Proceedings Series 5345; SPIE: Bellingham, WA, 2003; pp 194-205.
(9) Magennis, S. W.; Graham, E. M.; Jones, A. C. Agnew. Chem., Int. Ed. 2005,
44, 6512-6516.
(18) Benninger, R. K. P.; Hofmann, O.; McGinty, J.; Requejo-Isidro, J.; Munro,
I.; Neil, M. A. A.; deMello, A. J.; French, P. M. W. Opt. Express 2005, 13,
6275-6285.
(19) Elder, A. D.; Matthews, S. M.; Swartling, J.; Yunus, K.; Frank, J. H.; Brennan,
C. M.; Fisher, A. C.; Kaminski, C. F. Opt. Express 2006, 14, 5456-5467.
(20) Elder, A. D.; Frank, J. H.; Swartling, J.; Dai, X.; Kaminski, C. F. J. Microsc.
In press.
(21) Matthews, S. M.; Du, G. Q.; Fisher, A. C. J. Solid State Electrochem. 2006,
10 817-826.
10.1021/ac070045j CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/02/2007
Analytical Chemistry, Vol. 79, No. 11, June 1, 2007 4101