Single molecule detection in microfluidic chips for the analysis of cell signalling pathways

Microfluidic Antibody Capture (MAC) chips are small devices capable of quantifying biomarkers in single cells. These devices offer an all-optical approach for cell manipulation, lysis and single molecule quantification of a specific protein. This thesis details various developments to this device, both in terms of throughput and improvements to the single molecule counting process. The tumour suppressor protein p53 is a central hub for cellular stresses such as DNA damage, overproliferation and ribosomal biogenesis stress. Under stressed conditions p53 brings about the expression of a host of downstream effectors ultimately leading to DNA repair, temporary cell cycle arrest, senescence or apoptosis. The specifics of how p53 can lead to a number of different cell fate decisions are still unknown and require the development of quantitative biochemical techniques. In this thesis MAC chips are used to quantify p53 in single cells under a number of conditions. The chip data is used to create a quantitative model of p53 expression. This involved the use of stochastic simulation techniques such as the Gillespie algorithm and Approximate Bayesian Computation (ABC). These simulations determined that differences in p53 expression are best described as changes in the p53 degradation rate. This agrees with previous reports describing the p53-MDM2 relationship and its associated negative feedback loop. Lastly, attempts were made to obtain absolutely quantifiable data from the MAC chip platform. This involved calibrating the platform with known amounts of recombinant p53. By providing absolutely quantifiable data to the model of p53 expression the simulations could potentially provide real, biologically relevant parameters.