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

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.