This paper details the making, characterization, and use of a simple and versatile capillary-based co-axial
single-molecule mixing device which has a response time of 5-10 milliseconds and which can be used to monitor
bioconformational reactions and/or transient conformational states under non-equilibrium reactions conditions with
single molecule resolution. The device's co-axial geometry allows three-dimensional hydrodynamic focusing of sample
fluids to diffraction-limited dimensions where diffusional mixing is rapid and efficient. Its capillary-based design enables
rapid in-lab construction of mixers without the need for expensive lithography-based microfabrication facilities. In-line
filtering of sample fluids using granulated silica particles virtually eliminates clogging and extends the lifetime of each
device to many months. A major technical challenge dealt with here is the translation of spatial distances from the
mixing region into time-points for kinetic analyses. In order to obtain the required distance-to-time transfer and
instrument response functions for the device we characterize its fluid flow and mixing properties using both
Fluorescence Cross-Correlation Spectroscopy (FCCS) velocimetry and computational fluid dynamics (CFD) simulations.
We then apply the mixer to single molecule FRET protein folding studies of Chymotrypsin Inhibitor protein 2. By
transiently populating the unfolded state of CI2 under non-equilibrium in-vitro re-folding conditions, we spatially and
temporally resolve the denaturant-dependent non-specific collapse of the unfolded state from the barrier-limited folding
transition of CI2.