Microfluidic platform for integrated compartmentalization of single zoospores, germination and measurement of protrusive force generated by germ tubes

Abstract

This paper describes the design, fabrication and characterisation of a novel monolithic lab-on-a-chip (LOC) platform combining the trapping and germination of individual zoospores of the oomycete Achlya bisexualis with elastomeric micropillar-based protrusive force sensing. The oomycetes are of significant interest due to their pathogenic capabilities, which can have profound ecological and economic impacts. Zoospore encystment and germination via a germ tube play a key role in their pathogenicity. Our platform enables the study of these processes at a single cell level through hydrodynamic trapping of zoospores and their individual compartmentalization via normally closed pneumatic membrane microvalves. Valve geometry was optimized and media exchange characterized during dynamic valve operations to enhance the capture-to-growth ratio. We demonstrate germination of A. bisexualis zoospores on the platform and report three distinct germination patterns. Once germinated, germ tubes grew down growth channels towards single elastomeric micropillars. Tracking of pillar movement allowed for the measurement of microNewton range protrusive forces imparted by the tips of the germ tubes. Results indicate that the forces generated by the germ tubes are smaller than those exerted by mature hyphae. Through the use of parallel traps, channels and pillars on the same device, the platform enables high-throughput screening (HTS) of zoospores and their generation of protrusive force, an essential component of their infective capability. Due to its versatility, it will also allow for the screening of naturally bioactive compounds and the development of new biocontrol strategies for oomycetes, and morphologically similar fungal infections, as an alternative to agrochemicals.