Hyphae-on-a-chip : a microfluidic platform for the study of zoospore germination and protrusive forces in hyphal invasion.
| dc.contributor.author | Sun, Yiling | |
| dc.date.accessioned | 2020-07-07T00:56:10Z | |
| dc.date.available | 2020-07-07T00:56:10Z | |
| dc.date.issued | 2020 | en |
| dc.description.abstract | Fungal and oomycete pathogens have a significant influence on species extinctions, food security, ecosystem disturbances, and even human health. The large diversity of pathogenic fungi and oomycetes, and their acquired resistance to most antifungal agents, make treatment extremely difficult. Therefore, the aim of this work was to develop a lab-on-a-chip platform for high-throughput screening on individual spores and hyphae of fungi and oomycetes. This will help investigating and understanding the mechanisms of their invasive growth, and the development of antifungal compounds to control them. The ability of invasive growth of fungal and oomycete hyphae to penetrate through host tissue is essential for pathogenicity. Given the importance of protrusive force, which is considered as the force generated by hyphae to grow invasively, this thesis introduces two Lab-on-a-Chip (LOC) platforms with elastomeric micropillars as force sensors for the study of underlying mechanisms enabling force generation. In the first case, an existing mycelial LOC platform was improved to contain single free-bending micropillars in channel constrictions, which enabled the measurement of protrusive forces exerted by individual hyphal tips of fungi and oomycetes. The platform design, fabrication process and photoresist optimization required to adapt the microfluidic platform to different hyphae sizes and corresponding high aspect-ratio micropillars are reported. To demonstrate the applicability of the platform, oomycete Achlya bisexualis and fungus Neurospora crassa were cultured on the devices and the forces exerted by individual hyphae measured. For the second case, this thesis described the development of a novel monolithic LOC platform enabling high-throughput screening of different lifecycle stages and parameters of fungi and oomycetes, including spore germination, growth of resulting hyphae and their protrusive force generation. The platform integrates single zoospore capture and culture function with micropillar force sensing, allowing for investigations on an individual organism level. This is achieved by introducing hydrodynamic trapping of single cells and pneumatic membrane valves for compartmentalization. Single zoospores of oomycete A. bisexualis were demonstrated to be successfully captured in the trap sites via constriction structures in the parallel measurement channels, and the trapping and germination efficacies of two types of constrictions were tested. Two types of pneumatic membrane valves, normally-open and normally-closed microvalves, were implemented and evaluated for quality of compartmentalization in this thesis. Normally-closed microvalves with individual control for each measurement channel showed more effective single spore capture and compartmentalization. Using these valves, germinated hyphae from trapped oomycete A. bisexualis zoospores were observed to extend along measurement channels of the LOC platform, impacting with the force sensing micropillars, allowing for their growth rate and protrusive forces to be evaluated. In addition to the two LOC platforms, this thesis presents a number of other improvements on and contributions to device fabrication and experimentation, including high-resolution alignment marks for two-layer photoresist master; PDMS chip alignment and assembly for producing platforms with membrane valves, especially normally-closed microvalves; experimental setup for independent and precise control of channels with liquid or air, and in-depth characterization of flow on the platform during operation of normally-closed microvalves. | en |
| dc.identifier.uri | https://hdl.handle.net/10092/100708 | |
| dc.identifier.uri | http://dx.doi.org/10.26021/1430 | |
| dc.language | English | |
| dc.language.iso | en | |
| dc.publisher | University of Canterbury | en |
| dc.rights | All Right Reserved | en |
| dc.rights.uri | https://canterbury.libguides.com/rights/theses | en |
| dc.title | Hyphae-on-a-chip : a microfluidic platform for the study of zoospore germination and protrusive forces in hyphal invasion. | en |
| dc.type | Theses / Dissertations | en |
| thesis.degree.discipline | Electrical Engineering | en |
| thesis.degree.grantor | University of Canterbury | en |
| thesis.degree.level | Doctoral | en |
| thesis.degree.name | Doctor of Philosophy | en |
| uc.bibnumber | 2941702 | |
| uc.college | Faculty of Engineering | en |