Rapid Microfluidic Double-Jump Mixing Device for Single-Molecule Spectroscopy.

We introduce a microfluidic double-jump mixing device for investigating rapid biomolecular kinetics with confocal single-molecule spectroscopy. This device enables nonequilibrium dynamics to be probed, e.g., transiently populated intermediates that are inaccessible with existing single-molecule approaches. We demonstrate the potential and reliability of the method on time scales from milliseconds to minutes by investigating the coupled folding and binding reaction of two intrinsically disordered proteins and the conformational changes occurring in a large cytolytic pore-forming toxin.