Free-Flow Counterflow Gradient Focusing for Protein Fractionation
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In free-flow electrophoresis (FFE), a pressure-driven flow applied perpendicular to the electric field enables the continuous separation of charged solutes. Meanwhile, outlets located at the end of the separation region provide the opportunity for convenient sample collection. Consequently, FFE has drawn interest for many biological applications, including protein purification, real-time monitoring, and sample preparation prior to mass spectroscopy. However, the poor separation resolution associated with FFE has limited its potential. While advanced separation modes like free-flow isoelectric focusing (FF-IEF) offer improved performance, they often lead to further complications. The objective of this work is to introduce an alternative separation mode in FFE that can address some of its most prevalent limitations. This technique was inspired by a counterflow gradient focusing mechanism that has been studied extensively in capillary electrophoresis. In its present form, free-flow counterflow gradient focusing (FF-CGF) relies on an additional pressure-driven flow through the sidewalls of an FFE separation chamber. The added flow will then move towards the outlets, and in the process, create a velocity gradient in the direction of the electric field. As a result, charged solutes migrating across the separation region will focus at a position where their electrophoretic velocity is counterbalanced by the fluid flow. This mechanism is similar in principle to FF-IEF, except it does not require a complex separation medium, and focused solutes are less prone to precipitation. The first component of this work involves the physical modeling of the FF-CGF system. First, analytical equations describing the fluid velocity and the resulting focusing mechanism are derived. Then, a numerical model is provided for a detailed understanding of the solute dispersion that occurs during focusing. In particular, the interplay between molecular diffusion, the parabolic velocity profile of the counterflow, and the strength of the gradient is studied. An asymmetric solute distribution is demonstrated for a wide range of experimental conditions, and this knowledge can be used to predict separation resolution, throughput, and cross-contamination. Finally, the analytical and numerical models are updated to account for the presence of electroosmotic flow, which is shown to produce a pressure-driven backflow that can influence solute dispersion. The second component of this work involves the practical implementation of the FF-CGF system, and there is an emphasis on strategies for generating the counterflow gradient. The initial prototype is made using soft lithography, and high resistance side channels are used to produce a uniform counterflow gradient along the length of the chamber. The second prototype is made from a more rigid material, and provides a design that is more reliable and scalable. Furthermore, a commercial porous membrane is used to supply the counterflow. During this design, detailed guidelines are provided for the factors influencing device performance, including the porous parameters, the separation chamber dimensions, the outlet channels, and the ion exchange membranes needed to supply the electric field. Finally, a third prototype is constructed based on the aforementioned guidelines, and it is designed specifically for applications that require minute sample volumes. Moreover, this design can provide a better voltage efficiency, and operate at higher gradient strengths than the previous prototypes. Throughout this entire design process, the prototypes are used to demonstrate the separation of model proteins, and in general, the results are consistent with the analytical and numerical models. In the end, the design and implementation of FF-CGF highlights the versatility of this method, and may allow FFE to reach its full potential as a powerful protein fractionation tool.
Cite this version of the work
Matthew Courtney (2022). Free-Flow Counterflow Gradient Focusing for Protein Fractionation. UWSpace. http://hdl.handle.net/10012/18785