Design of droplet-based microfluidic devices and its application for protein fractionation

Abstract

Microfluidics technology offers new possibilities due to the nature of small scale, such as the high surface area to volume ratio. Droplet based microfluidics is an emerging field that has found its application in different areas, like material science, chemical reactions, and biochemical analysis. On the other hand, downstream processing operations like separation and purification still has some drawbacks like big reagents consumption, huge waste generation, extra steps like concentration or salts removal. Here is where microfluidics can be implemented to overcome some of the issues presented in this field. In this work, we designed and developed a microfluidic platform able to generate droplet-like aqueous two- phases system (ATPS) in a continuous manner. We started this project by performing a study of how different forces like surface tension, inertial forces, viscous forces, and geometry affect droplet formation. We found that all the previously mentioned parameters have an impact in different ways on the process of droplet formation. Afterwards, this knowledge was used to implement a microfluidic flow-focusing on the generation of droplet-based micro ATPS with the used of salts and PEG as the two liquid phases of the ATPS. To see that this ATPS droplet microfluidic system really worked, we tested its capacity to fractionate ribonuclease A (RNase A) on its native and PEGylated forms, which have affinity for the salt and PEG phases, respectively. Thus, we expected that proteins would get inside or outside droplets or streamlines according to their affinity for each of the two phases. In doing so, native, and PEGylated proteins were labeled with FITC and their movement from one to another phase or their retention in the phase where protein was placed were recorded. It was confirmed that proteins move or keep in their phase for which they have more affinity. This result opens the possibility for the development of microfluidic separators based on ATPS droplet formation.