A microfluidic platform to study pathogen-host interactions at single cell level [thesis]

Rui Zhang
In this study, a high throughput poly(dimethylsiloxane) (PDMS) array chip consisting of multiple micro-wells and microfluidic channels was developed to analyze two biological perspectives of pathogen-host interactions -adherence and gene transcription -at single cell level, that current techniques are unable to cater to. The association of a common human pathogen, Pseudomonas aeruginosa, in either single or mixed (with Staphylococcus aureus) infection context, to the host human lung epithelial
more » ... 549 cells was selected as the in vitro model to evaluate the performance of the chip. Single A549 cells were isolated into the individual micro-wells by one-step vacuum driven microfluidics when cell density was pre-adjusted to less than 0.3 cell/well. On-chip quantitative PCR was carried out with species-specific primer and probe sets to quantify the adhered bacteria. Single bacterium detection with the success rates of 90% for P. aeruginosa and 94% for S. aureus can be achieved with good reproducibility using the optimized DNA isolation protocol. Association profiling of P. aeruginosa and S. aureus in single infection context to A549 cells at three time points revealed different adherence patterns of these two pathogens. The attachment profiling of P. aeruginosa and S. aureus in mixed infection context was also obtained by on-chip multiplex q-PCR assay, from which P. aeruginosa association to the host A549 cells was identified to be significantly inhibited in the presence of S. aureus at 4 hours and 6 hours of infection. This chip was further developed for gene transcriptional regulation analysis by incorporation of a novel microfluidic phase partitioning technology for bacterial nucleic acid purification. DNA or RNA from P. aeruginosa and S. aureus in the range of 5000 down to a single cell in the sample volume of 1 µl or 125 nl, can be selectively recovered and directly put through on-chip quantitative PCR assay. The aqueous phase bacterial lysate was isolated in an array of micro-wells, after which an immiscible organic (phenolchloroform) phase was introduced in a headspace channel connecting the micro-well array. Continuous flow of the organic phase increases the interfacial contact with the aqueous phase to achieve purification of target nucleic acid through phase partitioning. Significantly enhanced nucleic acid recovery yield, up to 10 fold higher, was achieved
doi:10.32657/10356/54658 fatcat:gjqam35govd3lepunebleakq3a