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Microfluidic mixer designed for performing single-molecule kinetics with confocal detection on timescales from milliseconds to minutes

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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Figure 1: Microfluidic mixing device allowing observation on timescales from milliseconds to minutes.
Figure 2: Cartridge and cartridge holder for mounting the microfluidic devices on the single-molecule instrument.
Figure 3: Calibration curve for the temperature control of the microfluidic device.
Figure 4
Figure 5: Casting the PDMS devices.
Figure 6: Processing the PDMS cast.
Figure 7: Assembly of the microfluidic device.
Figure 8: Loading the cartridge holder.
Figure 9: Wide-field microscopy images of the mixing device in operation.
Figure 10: Normalized FCS curves, G(τ), of fluorescently labeled protein passing through the confocal volume positioned in the observation channel.
Figure 11: Unfolding kinetics of BdpA upon mixing with denaturant.
Figure 12: Kinetic measurement of the conformational change of ClyA upon mixing with DDM.

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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.).

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding authors

Correspondence to Daniel Nettels or Benjamin Schuler.

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The authors declare no competing financial interests.

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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