COPD is the third most prevalent disease globally and is poorly diagnosed. Methods to better understand the proteomic markers and disease progression are highly sought after but pose a technical challenge. Current standards are relatively rudimentary and will often overlap with other diseases or environmental factors such as smoking. Analysis at the proteomic level is challenging and collection of patient samples is typically highly invasive, and the current tests provide poor levels of data economy. The ability to ask proteomic questions on any disease state at a highly accurate level is a key tool to develop for a greater understanding of these diseases. Furthermore, the ability to use non-invasively derived patient samples, and obtain reliable data, opens new avenues for rapid and safe patient analysis.
The MAC chip is quantitative single molecule sensitive protein assay which can accurately evaluate absolute protein copy number in single cells. This thesis shows the design of two new assays to analyse the FoxO1 transcription factor total copy number and active copy number subpopulation. The works shows the two assays being validated on 3 cell lines. Followed with optimisations to the assays mid-way through the project to ensure they are robust and reliable for use with patient samples. Building on the creation of these novel assays, the assays are multiplexed within the MAC chip device to provide in tandem readouts of total and active FoxO1 copy number in two patient sample types. This thesis demonstrates the use of the assays on lung resection tissues and then on non-invasive sputum samples. The sputum samples demonstrate the ability of the MAC chip to isolate specific cell types with 100% accuracy from highly heterogenous samples. The use of patient samples in general shows the feasibility of utilising highly accurate and data rich single cell techniques with clinical materials.