A Droplet-Based Microfluidic Platform for the Production, Analysis and Use of Designer Proteinosomes

Compartmentalization represents a key step in the design and synthesis of artificial cell-like structures, effectively encapsulating molecules or entire reaction system in a semipermeable membrane thus isolating it from the surrounding environment. Typically such compartments are created through bulk emulsification methods, i.e. mechanical agitation, resulting in very broad size distribution which in turn may limit downstream reactions dependent on compartment size, e.g. sample encapsulation or mass transfer. Microfluidic droplet production methods developed over the last fifteen years may offer an alluring alternative to these bulk methodologies, resulting in better control over compartment size, composition and content. Herein we thus present a high-throughput droplet-based microfluidic platform for the production, analysis and manipulation of designer proteinosomes, artificial cells stabilized by a membrane composed of protein-polymer nanoconjugates. Initially an in-depth assessment of the latest developments in the field of synthetic biology is given, with special emphasis on microfluidic methodologies. This is followed by a description of the developed microfluidic devices and the design process and rational employed. Detailed protocols for all fabrication steps, as well as all experimental procedures are given. Utilizing the final device developed over the course of the PhD work presented significantly higher proteinosome monodispersity is achieved, and proteinosomes may be produced at significantly higher throughput compared to generally used bulk methodologies. Practical use of the generated proteinosomes is shown through functionalization of the nanoconjugate membrane with the enzyme glucose oxidase, which together with the encapsulated horseradish peroxidase forms a two-step reaction system. Microfluidically formed proteinosomes showed enhanced glucose oxidase/horseradish peroxidase activity compared to proteinosomes formed in bulk, further demonstrating our methods capabilities. Additionally, inspired by the way in which cells process information and make decisions we encapsulated a PEN-DNA toolbox based autocatalytic reaction system in proteinosomes. Components of the reaction system are initially encapsulated in semipermeable compartments and the proteinosomes transferred into an aqueous environment to which a stimulus can be added. Once the reaction is triggered it will propagate itself and spread to all droplets of a population mimicking the way in which soluble cues are detected and spread within a cell population. Finally strategies for the production of more complex artificial cells were investigated in a series of proof-of-concept experiments. The aim here was the generation of larger artificial structures mimicking cells, containing smaller compartments mimicking the function of cell organelles, with each artificial organelle performing a reaction part of a larger reaction system. Artificial organelles were generated using the methods described in previous chapters, and were then encapsulated in a larger proteinosome using a dedicated co-encapsulation device. These experiments, while delivering promising results are still limited in throughput, with improvements however this strategy may become an excellent production method for complex hierarchical reaction system, crucial for the advancement of the field of synthetic biology. In summary we presented a novel high-throughput production method for protein-polymer conjugate based artificial cells, resolving many of the issues faced to date using bulk production methods. We further demonstrated proteinosome functionalization and integration in simple reaction systems mimicking basic biological function. This may be expanded in the future to a large variety of biological reaction systems with increasing complexity, through both compartment functionalization as well as creation of hierarchical structures. Proteinosomes assembled in complex 3D orientations may also serve as artificial tissue systems. In addition to these more scientifical applications proteinosomes may present useful drug delivery applications due to their inherent biocompatibility, which may be further tailored and increased through a variety of surface modifications