Abstract
Probing the in vitro kinetics and dynamics of macromolecules involved in biological processes is important for discerning their mechanism and function. These dynamics span the sub-microsecond to millisecond and longer timescales. In addition to resolving dynamics and kinetics, structural characterization of non-equilibrium intermediates over these time scales is often desired. In this chapter, we review recent advances in microfluidic mixing methods (both low-Reynolds laminar and chaotic/turbulent mixers) for initiating biochemical reactions and provide an overview of the interfacing of these techniques with time-correlated single photon counting (TCSPC) fluorescent detection methods. We focus on approaches in which both a kinetic reaction time axis and a TCSPC time axis are simultaneously monitored, often referred to as a “double kinetic” experiment. Methods for measurement and analysis of these experiments are presented in which the TCSPC time axis corresponds to fluorescence lifetimes, time-resolved FRET or time-resolved anisotropy. An overview of matrix methods, such as singular value decomposition, and maximum entropy methods for data analysis are also reviewed.
Keywords
- Maximum Entropy Method
- Pulse Arrival Time
- Distance Distribution Function
- Fret Efficiency
- Microfluidic Mixer
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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Acknowledgments
The authors would like to thank McKenzie Davies for sharing representative data for the TCSPC analysis software figure, Brian Mackness and Jill Zitzewitz for helpful discussions and editing, and Bob Matthews for helpful discussions and support. This work was supported by NSF grants DBI1353942 to O. Bilsel and J.B. Perot and MCB1121942 to C.R. Matthews and O. Bilsel and NIH grant GM23303 to C.R. Matthews and O. Bilsel.
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Kathuria, S.V., Bilsel, O. (2015). Probing Microsecond Reactions with Microfluidic Mixers and TCSPC. In: Becker, W. (eds) Advanced Time-Correlated Single Photon Counting Applications. Springer Series in Chemical Physics, vol 111. Springer, Cham. https://doi.org/10.1007/978-3-319-14929-5_11
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