Abstract
Microfluidic mixing in combination with single-molecule spectroscopy allows the investigation of complex biomolecular processes under non-equilibrium conditions. Here we present a protocol for building, installing and operating microfluidic mixing devices optimized for this purpose. The mixer is fabricated by replica molding with polydimethylsiloxane (PDMS), which allows the production of large numbers of devices at a low cost using a single microfabricated silicon mold. The design is based on hydrodynamic focusing combined with diffusive mixing and allows single-molecule kinetics to be recorded over five orders of magnitude in time, from 1 ms to ∼100 s. Owing to microfabricated particle filters incorporated in the inlet channels, the devices provide stable flow for many hours to days without channel blockage, which allows reliable collection of high-quality data. Modular design enables rapid exchange of samples and mixing devices, which are mounted in a specifically designed holder for use with a confocal microscopy detection system. Integrated Peltier elements provide temperature control from 4 to 37 °C. The protocol includes the fabrication of a silicon master, production of the microfluidic devices, instrumentation setup and data acquisition. Once a silicon master is available, devices can be produced and experiments started within ∼1 d of preparation. We demonstrate the performance of the system with single-molecule Förster resonance energy transfer (FRET) measurements of kinetics of protein folding and conformational changes. The dead time of 1 ms, as predicted from finite element calculations, was confirmed by the measurements.
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References
Selvin, P.R. & Ha, T. Single-Molecule Techniques: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2008).
Dunkle, J.A. & Cate, J.H.D. Ribosome structure and dynamics during translocation and termination. Annual Review of Biophysics 39, 227–244 (2010).
Marshall, R.A., Aitken, C.E., Dorywalska, M. & Puglisi, J.D. Translation at the single-molecule level. Annu. Rev. Biochem. 77, 177–203 (2008).
Greenleaf, W.J., Woodside, M.T. & Block, S.M. High-resolution, single-molecule measurements of biomolecular motion. Annu. Rev. Biophys. Biomol. Struct. 36, 171–190 (2007).
Kapanidis, A.N. & Strick, T. Biology, one molecule at a time. Trends Biochem. Sci. 34, 234–243 (2009).
Ha, T., Kozlov, A.G. & Lohman, T.M. Single-molecule views of protein movement on single-stranded DNA. Annu. Rev. Biophys. 41, 295–319 (2012).
Smiley, R.D. & Hammes, G.G. Single-molecule studies of enzyme mechanisms. Chem. Rev. 106, 3080–3094 (2006).
Dittrich, P.S., Muller, B. & Schwille, P. Studying reaction kinetics by simultaneous FRET and cross-correlation analysis in a miniaturized continuous-flow reactor. Phys. Chem. Chem. Phys. 6, 4416–4420 (2004).
Zhuang, X.W. Single-molecule RNA science. in Annu. Rev. Biophys. Biomol. Struct. 34, 399–414 (2005).
Schuler, B. & Hofmann, H. Single-molecule spectroscopy of protein folding dynamics-expanding scope and timescales. Curr. Opin. Struct. Biol. 23, 36–47 (2013).
Nettels, D., Gopich, I.V., Hoffmann, A. & Schuler, B. Ultrafast dynamics of protein collapse from single-molecule photon statistics. Proc. Natl. Acad. Sci. USA 104, 2655–2660 (2007).
Soranno, A. et al. Quantifying internal friction in unfolded and intrinsically disordered proteins with single-molecule spectroscopy. Proc. Natl. Acad. Sci. USA 109, 17800–17806 (2012).
Neuweiler, H., Johnson, C.M. & Fersht, A.R. Direct observation of ultrafast folding and denatured state dynamics in single protein molecules. Proc. Natl. Acad. Sci. USA 106, 18569–74 (2009).
Sherman, E. & Haran, G. Fluorescence correlation spectroscopy of fast chain dynamics within denatured protein L. Chemphyschem. 12, 696–703 (2011).
Chung, H.S., Cellmer, T., Louis, J.M. & Eaton, W.A. Measuring ultrafast protein folding rates from photon-by-photon analysis of single-molecule fluorescence trajectories. Chem. Phys. http://dx.doi.org/10.1016/j.chemphys.2012.08.005 (14 August 2012).
Schuler, B., Lipman, E.A. & Eaton, W.A. Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy. Nature 419, 743–747 (2002).
Hoffmann, A. et al. Quantifying heterogeneity and conformational dynamics from single-molecule FRET of diffusing molecules: recurrence analysis of single particles (RASP). Phys. Chem. Chem. Phys. 13, 1857–1871 (2011).
Chung, H.S. et al. Extracting rate coefficients from single-molecule photon trajectories and FRET efficiency histograms for a fast-folding protein. J. Phys. Chem. A 115, 3642–3656 (2011).
Gopich, I.V. & Szabo, A. FRET efficiency distributions of multistate single molecules. J. Phys. Chem. B 114, 15221–15226 (2010).
Chung, H.S., Louis, J.M. & Eaton, W.A. Experimental determination of upper bound for transition path times in protein folding from single-molecule photon-by-photon trajectories. Proc. Natl. Acad. Sci. USA 106, 11837–11844 (2009).
Chung, H.S., McHale, K., Louis, J.M. & Eaton, W.A. Single-molecule fluorescence experiments determine protein folding transition path times. Science 335, 981–984 (2012).
Rhoades, E., Gussakovsky, E. & Haran, G. Watching proteins fold one molecule at a time. Proc. Natl. Acad. Sci. USA 100, 3197–3202 (2003).
Rhoades, E., Cohen, M., Schuler, B. & Haran, G. Two-state folding observed in individual protein molecules. J. Am. Chem. Soc. 126, 14686–14687 (2004).
Pirchi, M. et al. Single-molecule fluorescence spectroscopy maps the folding landscape of a large protein. Nat. Commun. 2, 493 (2011).
Kuzmenkina, E.V., Heyes, C.D. & Nienhaus, G.U. Single-molecule Förster resonance energy transfer study of protein dynamics under denaturing conditions. Proc. Natl. Acad. Sci. USA 102, 15471–15476 (2005).
Hofmann, H. et al. Single-molecule spectroscopy of protein folding in a chaperonin cage. Proc. Natl. Acad. Sci. USA 107, 11793–11798 (2010).
Gambin, Y. et al. Visualizing a one-way protein encounter complex by ultrafast single-molecule mixing. Nat. Methods 8, 239–241 (2011).
Lipman, E.A., Schuler, B., Bakajin, O. & Eaton, W.A. Single-molecule measurement of protein folding kinetics. Science 301, 1233–1235 (2003).
Borgia, A. et al. Localizing internal friction along the reaction coordinate of protein folding by combining ensemble and single-molecule fluorescence spectroscopy. Nat. Commun. 2, 1195 (2012).
Hamadani, K.M. & Weiss, S. Nonequilibrium single-molecule protein folding in a coaxial mixer. Biophys. J. 95, 352–365 (2008).
Brody, J.P., Yager, P., Goldstein, R.E. & Austin, R.H. Biotechnology at low Reynolds numbers. Biophys. J. 71, 3430–3441 (1996).
Knight, J.B., Vishwanath, A., Brody, J.P. & Austin, R.H. Hydrodynamic focusing on a silicon chip: mixing nanoliters in microseconds. Phys. Rev. Lett. 80, 3863–3866 (1998).
Zimmerman, W.B.J. Multiphysics Modeling with Finite Element Methods, (World Scientific Publishing, 2006).
Hoffmann, A. et al. Mapping protein collapse with single-molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy. Proc. Natl. Acad. Sci. USA 104, 105–110 (2007).
Kane, A.S. et al. Microfluidic mixers for the investigation of rapid protein folding kinetics using synchrotron radiation circular dichroism spectroscopy. Anal. Chem. 80, 9534–9541 (2008).
Liu, K., Tian, Y., Burrows, S.M., Reif, R.D. & Pappas, D. Mapping vortex-like hydrodynamic flow in microfluidic networks using fluorescence correlation spectroscopy. Analytica. Chimica. Acta. 651, 85–90 (2009).
Benninger, R.K. et al. Quantitative 3D mapping of fluidic temperatures within microchannel networks using fluorescence lifetime imaging. Anal. Chem. 78, 2272–2278 (2006).
Pfeil, S.H., Wickersham, C.E., Hoffmann, A. & Lipman, E.A. A microfluidic mixing system for single-molecule measurements. Rev. Sci. Instrum. 80, 055105 (2009).
Lemke, E.A. et al. Microfluidic device for single-molecule experiments with enhanced photostability. J. Am. Chem. Soc. 131, 13610–13612 (2009).
Gambin, Y., Simonnet, C., VanDelinder, V., Deniz, A. & Groisman, A. Ultrafast microfluidic mixer with three-dimensional flow focusing for studies of biochemical kinetics. Lab Chip 10, 598–609 (2010).
McDonald, J.C. & Whitesides, G.M. Poly(dimethylsiloxane) as a material for fabricating microfluidic devices. Acc. Chem. Res. 35, 491–499 (2002).
Hofmann, H. et al. Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy. Proc. Natl. Acad. Sci. USA 109, 16155–16160 (2012).
Arbour, T.J. & Enderlein, J. Application of dual-focus fluorescence correlation spectroscopy to microfluidic flow-velocity measurement. Lab Chip 10, 1286–1292 (2010).
Roder, H., Maki, K. & Cheng, H. Early events in protein folding explored by rapid mixing methods. Chem. Rev. 106, 1836–1861 (2006).
Cooksey, G.A., Plant, A.L. & Atencia, J. A vacuum manifold for rapid world-to-chip connectivity of complex PDMS microdevices. Lab Chip 9, 1298–1300 (2009).
Nettels, D. et al. Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins. Proc. Natl. Acad. Sci. USA 106, 20740–20745 (2009).
Kawahara, K. & Tanford, C. Viscosity and density of aqueous solutions of urea and guanidine hydrochloride. J. Biol. Chem. 241, 3228–3232 (1966).
CRC Handbook of Chemistry and Physics, (CRC Press, 2013).
Sisamakis, E., Valeri, A., Kalinin, S., Rothwell, P.J. & Seidel, C.A.M. Accurate single-molecule FRET studies using multiparameter fluorescence detection. Methods Enzymol. 475, 455–514 (2010).
Schuler, B. Application of single-molecule Förster resonance energy transfer to protein folding. Methods Mol. Biol. 350, 115–138 (2007).
Gösch, M., Blom, H., Holm, J., Heino, T. & Rigler, R. Hydrodynamic flow profiling in microchannel structures by single-molecule fluorescence correlation spectroscopy. Anal. Chem. 72, 3260–3265 (2000).
Dimitriadis, G. et al. Microsecond folding dynamics of the F13W G29A mutant of the B domain of staphylococcal protein A by laser-induced temperature jump. Proc. Natl. Acad. Sci. USA 101, 3809–3814 (2004).
Arora, P., Oas, T.G. & Myers, J.K. Fast and faster: a designed variant of the B-domain of protein A folds in 3 microsec. Protein Sci. 13, 847–853 (2004).
Mueller, M., Grauschopf, U., Maier, T., Glockshuber, R. & Ban, N. The structure of a cytolytic α-helical toxin pore reveals its assembly mechanism. Nature 459, 726–730 (2009).
Eifler, N. et al. Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state. EMBO J. 25, 2652–2661 (2006).
Schuler, B., Müller-Späth, S., Soranno, A. & Nettels, D. Application of confocal single-molecule FRET to intrinsically disordered proteins. Methods Mol. Biol. 896, 21–45 (2012).
Dittrich, P.S. & Schwille, P. Spatial two-photon fluorescence cross-correlation spectroscopy for controlling molecular transport in microfluidic structures. Anal. Chem. 74, 4472–4479 (2002).
Taylor, G. Conditions under which dispersion of a solute in a stream of solvent can be used to measure molecular diffusion. Proc. Royal Soc. London A 225, 473–477 (1954).
Acknowledgements
We thank E. Lipman for many helpful suggestions and discussions that have led to the routine application of the microfluidic mixer to biomolecular dynamics. We thank S. Radford for labeled BdpA and R. Glockshuber and D. Roderer for discussion and for an expression plasmid for ClyA. We thank R. Kellner for help with photography and video recording, A. Schmid for technical support, A. Soranno for help with data analysis and A. Hoffmann for help in the initial stages of the project. We thank all users of the device in the group for their feedback and suggestions. Electron microscopy was performed with support of the Center for Microscopy and Image Analysis, University of Zurich. This work was supported by the Swiss National Science Foundation, the Swiss National Center of Competence in Research (NCCR) for Structural Biology and a Starting Grant of the European Research Council (to B.S.).
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B.W., D.N. and B.S. designed the research and wrote the manuscript with the help of the other authors. B.W. constructed the instrumentation and mixing device, performed the microfabrication and experiments, and established the practical procedures. B.W. and D.N. performed data analysis and finite element calculations. B.W., S.B., J.C. and H.H. performed the experiments with protein samples, and S.B. and D.N. contributed to the temperature-control calibration. S.W. contributed to the design of the machined parts. S.H.P. established large parts of the practical procedures and helped with the design and handling of the device.
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Supplementary information
Supplementary Video 1
Microfluidic device assembly (MOV 14641 kb)
Supplementary Video 2
Cartridge assembly and loading (MOV 11718 kb)
Supplementary Table 1
Position-to-time conversion (PDF 511 kb)
Supplementary Data 1
Mask layout (ZIP 1248 kb)
Supplementary Data 2
Casting dish technical drawings (PDF 1453 kb)
Supplementary Data 3
Casting dish CAD files (ZIP 1470 kb)
Supplementary Data 4
Cartridges for holding microfluidic devices and temperature controlled cartridge holder technical drawings (PDF 6317 kb)
Supplementary Data 5
Cartridges for holding microfluidic devices and temperature controlled cartridge holder CAD files (ZIP 4699 kb)
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Wunderlich, B., Nettels, D., Benke, S. et al. Microfluidic mixer designed for performing single-molecule kinetics with confocal detection on timescales from milliseconds to minutes. Nat Protoc 8, 1459–1474 (2013). https://doi.org/10.1038/nprot.2013.082
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DOI: https://doi.org/10.1038/nprot.2013.082
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