This has been particularly important in studies involving helicase ATP hydrolysis 2, 3, 5, where the ATP gradient leads to a gradual start of the translocation by the enzyme, with a slower velocity right after injection. Furthermore, the velocities employed not to perturb the system usually lead to the generation of a reagent gradient in the flow cell 3, 12. A fast exchange of reagents is not possible without working under very high flow rates, thus disturbing the biological system in the flow cell. While this system, comprising a single-channel flow cell and a syringe pump, is useful and precise enough in a vast variety of single-molecule assays 21, 22, 23, 24, 25, it has a main limitation. However, the standard experimental scenario maintains at least an inlet and an outlet open for reagent exchange 12, 19, 20. In some cases, flow cells are afterwards sealed to perform the experiment 16. This can be avoided by introducing reagents in a controlled manner using a syringe pump 3, 17, 18, which is the most common method. However, these methods can generate uncontrolled and turbulent flow-rates that could shear DNA tethers. Reagents can be then introduced in different ways including pipetting 15 or sucking with a piece of paper 16. The affordability of materials as well as the simple fabrication procedure makes them worthless to recycle, with the exception of those made of quartz slides 13, 14. These cells are normally limited to a number of experiments and are then discarded. Furthermore, these give versatility in the design and fabrication procedure. Commercial solutions are available for sophisticated applications, but custom-built flow cells are the most widespread approach. double-sided tape, which is thinner, or parafilm, which is thicker 12. The volume of the flow cell can be controlled by modifying the dimensions (length, width and depth) of the channel and the number of gasket layers, as well as the gasket material, i.e. The channel can be created using a gasket made of paraffin wax (parafilm) or double-sided tape (Fig. Simplest flow cells are normally home-fabricated using two coverslips or microscope slides (made of glass or quartz) with a channel in between.
helicases which need ATP 2, 3, 4, 5, or require sequential addition of proteins in complex DNA–protein interaction reactions involved in processes such as DNA replication, transcription, DNA repair or chromosome organization 6, 7, 8, 9, 10. This is crucial in reactions that need to be triggered by ligands, i.e. Additionally, it is useful to allow the exchange of reagents in the experiment. Single-molecule techniques such as magnetic tweezers (MT), optical tweezers (OT) or fluorescence microscopy employ flow cells to confine the biological molecules of interest in a reduced environment, normally under close to physiological buffer conditions 1. Finally, we study the potential applicability of the platform to study kinetics at the single molecule level. We additionally define a phenomenological expression to predict the boundary switching time for a particular flow cell cross section.
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. For very precise measurements it is thus desirable to have a very fast exchange of reagents with minimal diffusion. 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.
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. Home-fabricated flow cells using two glass coverslips and a gasket made of paraffin wax are a widespread approach. This is crucial in experiments that need to be triggered by ligands or require a sequential addition of proteins. 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. Single-molecule experiments usually take place in flow cells.