Characterizing microfluidic approaches for a fast and efficient reagent exchange in single-molecule studies

Single-molecule experiments usually take place in flow cells. This experimental approach is essential for experiments requiring a liquid environment, but is also useful to allow the exchange of reagents before or during measurements. This is crucial in experiments that need to be triggered by ligands or require a sequential addition of proteins. Home-fabricated flow cells using two glass coverslips and a gasket made of paraffin wax are a widespread approach. The volume of the flow cell can be controlled by modifying the dimensions of the channel while the reagents are introduced using a syringe pump. In this system, high flow rates disturb the biological system, whereas lower flow rates lead to the generation of a reagent gradient in the flow cell. For very precise measurements it is thus desirable to have a very fast exchange of reagents with minimal diffusion. We propose the implementation of multistream laminar microfluidic cells with two inlets and one outlet, which achieve a minimum fluid switching time of 0.25 s. We additionally define a phenomenological expression to predict the boundary switching time for a particular flow cell cross section. Finally, we study the potential applicability of the platform to study kinetics at the single molecule level.