| dc.description.abstract | Microorganisms in the environment is extremely diverse, yet those that are identified and their functions known are extremely small. Humans have been increasingly coming in contact with these diverse microorganisms stemming from increases in global transportation and urbanization. The emergence of readily available gene editing tools as well as continuing evolution of these microorganisms as they come in contact with human make the biocomplexity even higher. Together, the potential bacterial pathogens in these environments pose significant risk to the public. Methods and tools that can provide comprehensive, systematic, and rapid analyses of these microorganisms can greatly contribute to our understanding of these extremely complex microbial world, especially for microbial pathogens. The research presented here provides three new strategies: first, a proactive strategy to study and understand the molecular mechanisms by which pathogens emerge and evolve, and second, an effective surveillance system that is capable of detecting and analyzing the emerging pathogens in timely manner, and third, development of microfluidic technologies that can enable high-throughput investigation of biological samples. With these strategies in mind, this dissertation presents the development, testing, and utilization of several novel microfluidics systems that each allow different microbial interrogation approaches to be performed in a high-throughput lab-on-a-chip format for combating the emergence of microbial pathogens.
The first platform developed is a microfluidic system named SEER platform, System for Evaluating the Emergence of Replicating pathogens, which enables the fully automated, multi-round directed evolution of intracellular parasitism in the laboratory. The SEER platform utilizes a porous membrane filter-based selective cell manipulation microfluidic technology and can direct naïve bacterial populations that are initially incapable of intracellular bacterial parasitism to evolve and generate populations that can display enhanced survival within macrophages, so to determine genetic loci that confer this phenotype. This platform was successfully utilized to study the symbiotic evolutionary process between E. coli DH5α strain and RAW264.7 macrophage and have confirmed the contribution of cpxR gene on the enhanced survival phenotype. The second platform is a high-throughput, dielectrophoresis-based microfluidics platform that can achieve selective manipulation of cells from mixed cell communities in a non-destructive, single-cell resolution manner, which allows microorganisms that adhere to mammalian host cells to be selected and sorted for further analysis of their pathogenicity. This platform was successfully utilized to investigate two environmental soil samples and various adherent pathogens that originally presented in the soil with low abundancy were extracted and identified with this high-throughput microfluidic method.
In addition to the successful development of two microfluidic systems for host-pathogen interaction studies, several microfluidic technology advancements have also been achieved based on the utilization of dielectrophoresis phenomena. These include the development of in-droplet cell separation technology, in-droplet solution exchange technology, droplet size-based sorting technology, as well as new microfabrication architectures that can significantly improve the performances of microfluidic systems. All developed technologies have been successfully validated and their utilities demonstrated and are expected to greatly expand the potential application of microfluidic systems in conducting cell biology assays. | |
