Aqueous Phase Separation within Water-in-Oil Emulsions as Cellular Models
Restricted (Penn State Only)
- Author:
- Crowe, Charles
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 18, 2021
- Committee Members:
- Philip Bevilacqua, Program Head/Chair
Paul Cremer, Major Field Member
Peter Butler, Outside Unit & Field Member
Scott Showalter, Major Field Member
Christine Keating, Chair & Dissertation Advisor - Keywords:
- liquid-liquid phase separation
phase separation
microfluidics
neutral polymer phase separation
emulsion
water-in-oil
coacervation
bioinspired
artificial cell
microscopy
droplet - Abstract:
- The cellular environment is a crowded and compartmentalized place, where differing chemical microenvironments are able to coexist in solution through the use of membrane-bound and membraneless organelles. This biological complexity can be modeled through the use of liquid-liquid phase separation (LLPS), where macromolecular solutes drive the formation of coexisting aqueous phases. Further encapsulation of such multiphase systems within water-in-oil emulsions provides the opportunity to study these complex environments within an isolated system. This dissertation explores the physical chemistry implications of encapsulating these phase-separated solutions, focusing on the ability to understand and control specific solution characteristics. Chapter 1 provides an overview of the mechanisms behind various forms of LLPS and how the phenomena has been used in the production of artificial cells. Specifically, both associative and segregative phase separation are discussed, as well as the production of water-in-oil emulsions containing such solutions. Additionally, the technique of microfluidics is introduced, which subsequent chapters will be explored as a method of standardizing and controlling the production of droplet-based cellular mimics. Chapter 2 details the use of water-in-hydrocarbon emulsions containing a poly(ethylene glycol) (PEG) and dextran aqueous two-phase system (ATPS) as biomimetic systems to house transcription and translation. With RNA aptamer-fluorophore systems, the process of transcription was observed, followed by the production of a fluorescent protein through subsequent translation. Observations regarding the effect of water-in-oil encapsulation on such multiphase solutions in this chapter laid the foundations for the work that followed, highlighting the need for additional control over the resulting emulsions so that specific aspects of the systems could be investigated in an intentional manner. Chapter 3 describes the implementation of microfluidics to achieve this control over droplet microenvironment. ATPS were created within water-in-fluorocarbon emulsions, using the partitioning of fluorescently labeled PEG and dextran to determine the resulting phase compositions after droplet production via fluorescence microscopy. This allowed us to calculate the tie line length of each population of ATPS droplets created with specified microfluidic parameters, which could be rationally adjusted to control the subsequent partitioning of biomolecular probes between the aqueous phases. Chapter 4 discusses the formation of solid-like particles within both single-phase and phase-separated solutions containing dextran. This behavior is similar to the biological formation of solid aggregates within living systems that are often indications of disease. By exploring the effect of polymer concertation, phase separation, and emulsification on the formation of this material, we demonstrated that the key factor was the local concentration of dextran, with higher local concentration leading to faster material formation. Photobleaching experiments revealed the solid-like nature of the material, and the inclusion of biomolecular probes showed the impact of intermolecular interactions on the resulting partitioning. Chapter 5 considers the microfluidic production of water-in-fluorocarbon emulsions that include coexisting aqueous phases incorporating both associative and segregative phase separation. Protamine sulfate was used to form the associative phase-separated component, while a PEG/dextran ATPS comprised the segregative phase separation. The resulting emulsions exhibited behavior not observed with those containing only segregative phase separation, such as the ability to stabilize water-in-fluorocarbon droplets against coalescence without the presence of the previously required water-soluble surfactant. Chapter 6 outlines the general conclusions of this dissertation and suggests future work that builds upon the topics discussed here. Through the combination of liquid-liquid phase separation, water-in-oil emulsions, and microfluidics, the knowledge and methods detailed in this dissertation have expanded the set of tools available to researchers, enabling more precise and rational creation of cellular mimics with desired characteristics.