Skip to main content
SpringerLink
Log in

Probing Microsecond Reactions with Microfluidic Mixers and TCSPC

  • Chapter
  • First Online:
Advanced Time-Correlated Single Photon Counting Applications

Part of the book series: Springer Series in Chemical Physics ((CHEMICAL,volume 111))

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. K.N. Aprilakis, H. Taskent, D.P. Raleigh, Use of the novel fluorescent amino acid p-cyanophenylalanine offers a direct probe of hydrophobic core formation during the folding of the N-terminal domain of the ribosomal protein L9 and provides evidence for two-state folding. Biochemistry 46, 12308–12313 (2007)

    Article  CAS  Google Scholar 

  2. W. Becker, Advanced Time-Correlated Single-Photon Counting Techniques (Springer, Berlin, 2005)

    Book  Google Scholar 

  3. W. Becker, The bh TCSPC Handbook, 5th edn. Becker & Hickl GmbH (2012), www.becker-hickl.com, printed copies available from Becker & Hickl GmbH

  4. W. Becker, B. Su, K. Weisshart, O. Holub, FLIM and FCS detection in laser-scanning microscopes: increased efficiency by GaAsP hybrid detectors. Microsc. Res. Tech. 74, 804–811 (2011)

    CAS  Google Scholar 

  5. J.M. Beechem, E. Haas, Simultaneous determination of intramolecular distance distributions and conformational dynamics by global analysis of energy transfer measurements. Biophys. J. 55, 1225–1236 (1989)

    Article  CAS  Google Scholar 

  6. J.M. Beechem, Global analysis of biochemical and biophysical data. Methods Enzymol. 210, 37–54 (1992)

    Article  CAS  Google Scholar 

  7. J.M. Beechem, Picosecond fluorescence decay curves collected on millisecond time scale: direct measurement of hydrodynamic radii, local/global mobility, and intramolecular distances during protein-folding reactions. Methods Enzymol. 278, 24–49 (1997)

    Article  CAS  Google Scholar 

  8. Y. Bellouard, T. Colomb, C. Depeursinge, M. Dugan, A.A. Said, P. Bado, Nanoindentation and birefringence measurements on fused silica specimen exposed to low-energy femtosecond pulses. Opt. Express 14, 8360–8366 (2006)

    Article  CAS  Google Scholar 

  9. O. Bilsel, L. Yang, J.A. Zitzewitz, J.M. Beechem, C.R. Matthews, Time-resolved fluorescence anisotropy study of the refolding reaction of the alpha-subunit of tryptophan synthase reveals nonmonotonic behavior of the rotational correlation time. Biochemistry 38, 4177–4187 (1999)

    Article  CAS  Google Scholar 

  10. O. Bilsel, C. Kayatekin, L.A. Wallace, C.R. Matthews, A microchannel solution mixer for studying microsecond protein folding reactions. Rev. Sci. Instrum. 76, 014302 (2005)

    Article  Google Scholar 

  11. D.J. Birch, D. McLoskey, A. Sanderson, K. Suhling, A.S. Holmes, Multiplexed time-correlated single-photon counting. J Fluoresc 4, 91–102 (1994)

    Article  CAS  Google Scholar 

  12. J.C. Brochon, Maximum entropy method of data analysis in time-resolved spectroscopy. Methods Enzymol. 240, 262–311 (1994)

    Article  CAS  Google Scholar 

  13. Y. Chen, A. Periasamy, Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization. Microsc. Res. Tech. 63, 72–80 (2004)

    Article  CAS  Google Scholar 

  14. R.B. Dyer, E.B. Brauns, Laser-induced temperature jump infrared measurements of RNA folding. Methods Enzymol. 469, 353–372 (2009)

    Article  CAS  Google Scholar 

  15. W.A. Eaton, V. Munoz, S.J. Hagen, G.S. Jas, L.J. Lapidus, E.R. Henry, J. Hofrichter, Fast kinetics and mechanisms in protein folding. Annu. Rev. Biophys. Biomol. Struct. 29, 327–359 (2000)

    Article  CAS  Google Scholar 

  16. T. Egawa, J.L. Durand, E.Y. Hayden, D.L. Rousseau, S.R. Yeh, Design and evaluation of a passive alcove-based microfluidic mixer. Anal. Chem. 81, 1622–1627 (2009)

    Article  CAS  Google Scholar 

  17. E.L. Elson, Fluorescence correlation spectroscopy: past, present, future. Biophys. J. 101, 2855–2870 (2011)

    Article  CAS  Google Scholar 

  18. S. Felekyan, H. Sanabria, S. Kalinin, R. Kühnemuth, C.A. Seidel, Analyzing förster resonance energy transfer with fluctuation algorithms. Methods Enzymol. 519, 39–85 (2013)

    Article  CAS  Google Scholar 

  19. J.J. Fisz, Fluorescence polarization spectroscopy at combined high-aperture excitation and detection: application to one-photon-excitation fluorescence microscopy. J. Chem. Phys. A 111, 8606–8621 (2007)

    Article  CAS  Google Scholar 

  20. F. Gai, D. Du, Y. Xu, Infrared temperature-jump study of the folding dynamics of alpha-helices and beta-hairpins. Methods Mol. Biol. 350, 1–20 (2007)

    CAS  Google Scholar 

  21. Y. Gambin, C. Simonnet, V. VanDelinder, A. Deniz, A. Groisman, Ultrafast microfluidic mixer with three-dimensional flow focusing for studies of biochemical kinetics. Lab Chip 10, 598–609 (2010)

    Article  CAS  Google Scholar 

  22. Y. Gambin, V. VanDelinder, A.C. Ferreon, E.A. Lemke, A. Groisman, A.A. Deniz, Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing. Macmillan Publishers Ltd, Nat. Methods 8, 239–241 (2011)

    Google Scholar 

  23. M. Gentin, M. Vincent, J.C. Brochon, A.K. Livesey, N. Cittanova, J. Gallay, Time-resolved fluorescence of the single tryptophan residue in rat alpha-fetoprotein and rat serum albumin: analysis by the maximum-entropy method. Biochemistry 29, 10405–10412 (1990)

    Article  CAS  Google Scholar 

  24. R. Gilmanshin, S. Williams, R.H. Callender, W.H. Woodruff, R.B. Dyer, Fast events in protein folding: relaxation dynamics of secondary and tertiary structure in native apomyoglobin. Proc. Natl. Acad. Sci. U.S.A. 94, 3709–3713 (1997)

    Article  CAS  Google Scholar 

  25. E. Haas, Ensemble FRET methods in studies of intrinsically disordered proteins. Methods Mol. Biol. 895, 467–498 (2012)

    Article  CAS  Google Scholar 

  26. S.J. Hagen, J. Hofrichter, A. Szabo, W.A. Eaton, Diffusion-limited contact formation in unfolded cytochrome c: estimating the maximum rate of protein folding. Proc. Natl. Acad. Sci. U.S.A. 93, 11615–11617 (1996)

    Article  CAS  Google Scholar 

  27. K.M. Hamadani, S. Weiss, Nonequilibrium single molecule protein folding in a coaxial mixer. Biophys. J. 95, 352–365 (2008)

    Article  CAS  Google Scholar 

  28. D.F. Hansen, H. Feng, Z. Zhou, Y. Bai, L.E. Kay, Selective characterization of microsecond motions in proteins by NMR relaxation. J. Am. Chem. Soc. 131, 16257–16265 (2009)

    Article  CAS  Google Scholar 

  29. E.B. Ishay, G. Hazan, G. Rahamim, D. Amir, E. Haas, An instrument for fast acquisition of fluorescence decay curves at picosecond resolution designed for “double kinetics” experiments: application to fluorescence resonance excitation energy transfer study of protein folding. Rev. Sci. Instrum. 83, 084301 (2012)

    Article  Google Scholar 

  30. K.C. Jones, C.S. Peng, A. Tokmakoff, Folding of a heterogeneous beta-hairpin peptide from temperature-jump 2D IR spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 110, 2828–2833 (2013)

    Article  CAS  Google Scholar 

  31. S.V. Kathuria, L. Guo, R. Graceffa, R. Barrea, R.P. Nobrega, C.R. Matthews, T.C. Irving, O. Bilsel, Minireview: structural insights into early folding events using continuous-flow time-resolved small-angle X-ray scattering. Biopolymers 95, 550–558 (2011)

    Article  CAS  Google Scholar 

  32. S.V. Kathuria, C. Kayatekin, R. Barrea, E. Kondrashkina, R. Graceffa, L. Guo, R.P. Nobrega, S. Chakravarthy, C.R. Matthews, T.C. Irving, O. Bilsel, Microsecond barrier-limited chain collapse observed by time-resolved FRET and SAXS. J. Mol. Biol. 426(9), 1980–1994 (2014)

    Article  CAS  Google Scholar 

  33. S.V. Kathuria, A. Chan, R. Graceffa, R. Paul Nobrega, C. Robert Matthews, T.C. Irving, B. Perot, O. Bilsel, Advances in turbulent mixing techniques to study microsecond protein folding reactions. Biopolymers 99, 888–96 (2013)

    Google Scholar 

  34. B.A. Kelch, D.L. Makino, M. O’Donnell, J. Kuriyan, How a DNA polymerase clamp loader opens a sliding clamp. Science 334, 1675–1680 (2011)

    Article  CAS  Google Scholar 

  35. T. Kimura, J.C. Lee, H.B. Gray, J.R. Winkler, Site-specific collapse dynamics guide the formation of the cytochrome c’ four-helix bundle. Proc. Natl. Acad. Sci. U.S.A. 104, 117–122 (2007)

    Article  CAS  Google Scholar 

  36. M. Köllner, J. Wolfrum, How many photons are necessary for fluorescence-lifetime measurements? Phys. Chem. Lett. 200, 199–204 (1992)

    Article  Google Scholar 

  37. J. Kubelka, W.A. Eaton, J. Hofrichter, Experimental tests of villin subdomain folding simulations. J. Mol. Biol. 329, 625–630 (2003)

    Article  CAS  Google Scholar 

  38. A.T.N. Kumar, L. Zhu, J.F. Christian, A.A. Demidov, P.M. Champion, On the rate distribution analysis of kinetic data using the maximum entropy method: applications to myoglobin relaxation on the nanosecond and femtosecond timescales. J. Phys. Chem. B 105, 7847–7856 (2001)

    Article  CAS  Google Scholar 

  39. J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd edn. (Springer, Berlin, 2006)

    Book  Google Scholar 

  40. T.J. Lane, D. Shukla, K.A. Beauchamp, V.S. Pande, To milliseconds and beyond: challenges in the simulation of protein folding. Curr. Opin. Struct. Biol. 23, 58–65 (2013)

    Article  CAS  Google Scholar 

  41. L.J. Lapidus, S. Yao, K.S. McGarrity, D.E. Hertzog, E. Tubman, O. Bakajin, Protein hydrophobic collapse and early folding steps observed in a microfluidic mixer. Biophys. J. 93, 218–224 (2007)

    Article  CAS  Google Scholar 

  42. Y. Li, D. Zhang, X. Feng, Y. Xu, B.F. Liu, A microsecond microfluidic mixer for characterizing fast biochemical reactions. Talanta 88, 175–180 (2012)

    Article  CAS  Google Scholar 

  43. K. Lindorff-Larsen, S. Piana, R.O. Dror, D.E. Shaw, How fast-folding proteins fold. Science 334, 517–520 (2011)

    Article  CAS  Google Scholar 

  44. F. Liu, M. Nakaema, M. Gruebele, The transition state transit time of WW domain folding is controlled by energy landscape roughness. J. Chem. Phys. 131 (2009)

    Google Scholar 

  45. A.K. Livesey, J.C. Brochon, Analyzing the distribution of decay constants in pulse-fluorimetry using the maximum entropy method. Biophys. J. 52, 693–706 (1987)

    Article  CAS  Google Scholar 

  46. K. Malecha, L.J. Golonka, J. Bałdyga, M. Jasińska, P. Sobieszuk, Serpentine microfluidic mixer made in LTCC. Sens. Actuators, B 143, 400–413 (2009)

    Article  Google Scholar 

  47. V. Munoz, P.A. Thompson, J. Hofrichter, W.A. Eaton, Folding dynamics and mechanism of beta-hairpin formation. Nature 390, 196–199 (1997)

    Article  CAS  Google Scholar 

  48. M.R. Otto, M.P. Lillo, J.M. Beechem, Resolution of multiphasic reactions by the combination of fluorescence total-intensity and anisotropy stopped-flow kinetic experiments. Biophys. J. 67, 2511–2521 (1994)

    Article  CAS  Google Scholar 

  49. S.A. Pabit, S.J. Hagen, Laminar-flow fluid mixer for fast fluorescence kinetics studies. Biophys. J. 83, 2872–2878 (2002)

    Article  CAS  Google Scholar 

  50. S.A. Pabit, J.L. Sutton, H. Chen, L. Pollack, Role of ion valence in the submillisecond collapse and folding of a small RNA domain. Biochemistry 52, 1539–1546 (2013)

    Article  CAS  Google Scholar 

  51. A.G. Palmer, Nmr probes of molecular dynamics: overview and comparison with other techniques. Annu. Rev. Biophys. Biomol. Struct. 30, 129–155 (2001)

    Article  CAS  Google Scholar 

  52. H.Y. Park, X. Qiu, E. Rhoades, J. Korlach, L.W. Kwok, W.R. Zipfel, W.W. Webb, L. Pollack, Achieving uniform mixing in a microfluidic device: hydrodynamic focusing prior to mixing. Anal. Chem. 78, 4465–4473 (2006)

    Article  CAS  Google Scholar 

  53. C.M. Phillips, Y. Mizutani, R.M. Hochstrasser, Ultrafast thermally induced unfolding of RNase A. Proc. Natl. Acad. Sci. U.S.A. 92, 7292–7296 (1995)

    Article  CAS  Google Scholar 

  54. L. Pollack, M.W. Tate, N.C. Darnton, J.B. Knight, S.M. Gruner, W.A. Eaton, R.H. Austin, Compactness of the denatured state of a fast-folding protein measured by submillisecond small-angle x-ray scattering. Proc. Natl. Acad. Sci. U.S.A. 96, 10115–10117 (1999)

    Article  CAS  Google Scholar 

  55. V. Ratner, D. Amir, E. Kahana, E. Haas, Fast collapse but slow formation of secondary structure elements in the refolding transition of E. coli adenylate kinase. J. Mol. Biol. 352, 683–699 (2005)

    Article  CAS  Google Scholar 

  56. P. Regenfuss, R.M. Clegg, M.J. Fulwyler, F.J. Barrantes, T.M. Jovin, Mixing liquids in microseconds. Rev. Sci. Instrum. 56, 283 (1985)

    Article  CAS  Google Scholar 

  57. H. Roder, K. Maki, H. Cheng, Early events in protein folding explored by rapid mixing methods. Chem. Rev. 106, 1836–1861 (2006)

    Article  CAS  Google Scholar 

  58. J.M. Rogers, L.G. Lippert, F. Gai, Non-natural amino acid fluorophores for one- and two-step fluorescence resonance energy transfer applications. Anal. Biochem. 399, 182–189 (2010)

    Article  CAS  Google Scholar 

  59. F. Schonfeld, V. Hessel, C. Hofmann, An optimised split-and-recombine micro-mixer with uniform ‘chaotic’ mixing. Lab Chip 4, 65–69 (2004)

    Article  CAS  Google Scholar 

  60. M.C. Shastry, S.D. Luck, H. Roder, A continuous-flow capillary mixing method to monitor reactions on the microsecond time scale. Biophys. J. 74, 2714–2721 (1998)

    Article  CAS  Google Scholar 

  61. J. Skilling, R.K. Bryan, Maximum entropy image reconstruction: general algorithm. Mon. Not. R. Astron. Soc. 211, 111–124 (1984)

    Article  Google Scholar 

  62. T.R. Sosnick, D. Barrick, The folding of single domain proteins–have we reached a consensus? Curr. Opin. Struct. Biol. 21, 12–24 (2011)

    Article  CAS  Google Scholar 

  63. A.D. Stroock, S.K.W. Dertinger, A. Ajdari, I. Mezic, H.A. Stone, G.M. Whitesides, Chaotic mixer for microchannels. Science 295, 647–651 (2002)

    Article  CAS  Google Scholar 

  64. S. Takahashi, S.R. Yeh, T.K. Das, C.K. Chan, D.S. Gottfried, D.L. Rousseau, Folding of cytochrome c initiated by submillisecond mixing. Nat. Struct. Biol. 4, 44–50 (1997)

    Article  CAS  Google Scholar 

  65. H. Taskent-Sezgin, P. Marek, R. Thomas, D. Goldberg, J. Chung, I. Carrico, D.P. Raleigh, Modulation of p-cyanophenylalanine fluorescence by amino acid side chains and rational design of fluorescence probes of alpha-helix formation. Biochemistry 49, 6290–6295 (2010)

    Article  CAS  Google Scholar 

  66. D. Thirumalai, B.-Y. Ha, Statistical mechanics of semiflexible chains: a meanfield variational approach. arXiv:cond-mat/9705200 (1997)

    Google Scholar 

  67. P.A. Thompson, W.A. Eaton, J. Hofrichter, Laser temperature jump study of the helix <==> coil kinetics of an alanine peptide interpreted with a ‘kinetic zipper’ model. Biochemistry 36, 9200–9210 (1997)

    Article  CAS  Google Scholar 

  68. C.T. Wang, Y.C. Hu, T.Y. Hu, Biophysical micromixer. Sens. (Basel) 9, 5379–5389 (2009)

    Article  CAS  Google Scholar 

  69. Y. Wu, E. Kondrashkina, C. Kayatekin, C.R. Matthews, O. Bilsel, Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein. Proc. Natl. Acad. Sci. U.S.A. 105, 13367–13372 (2008)

    Article  CAS  Google Scholar 

  70. B. Wunderlich, D. Nettels, S. Benke, J. Clark, S. Weidner, H. Hofmann, S.H. Pfeil, B. Schuler, Microfluidic mixer designed for performing single-molecule kinetics with confocal detection on timescales from milliseconds to minutes. Macmillan Publishers Ltd, Nat. Protoc. 8, 1459–1474 (2013)

    Google Scholar 

  71. W.Y. Yang, M. Gruebele, Folding at the speed limit. Nature 423, 193–197 (2003)

    Article  CAS  Google Scholar 

  72. G. Zoldak, J. Stigler, B. Pelz, H. Li, M. Rief, Ultrafast folding kinetics and cooperativity of villin headpiece in single-molecule force spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 110, 18156–18161 (2013)

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, 01605, USA

    Sagar V. Kathuria & Osman Bilsel

Authors
  1. Sagar V. Kathuria
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Osman Bilsel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Osman Bilsel .

Editor information

Editors and Affiliations

  1. Becker & Hickl GmbH, Berlin, Germany

    Wolfgang Becker

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer International Publishing Switzerland

About this chapter

Cite this chapter

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

Download citation

Publish with us

Policies and ethics

Search

Navigation