Single cell analysis provides the ability to profile the heterogeneity in protein expression that exists within a population of cells, thereby providing a key insight into cellular function, disease progression and drug resistance. However, the field of single-cell proteomics is burdened with technical challenges, including the protein domain size, meaning many of these techniques do not have the sensitivity required to quantify proteins of low concentration in individual cells. The use of microfluidics devices for single cell proteomics presents numerous benefits, including the ability to quantify low protein counts from single cells, as well as low sample consumption.
The microfluidic affinity capture (MAC) chip is a microfluidic device capable of quantifying protein copy numbers from single cells. In this thesis, we detail the design, fabrication and experimental format of the MAC chip.
Chapter 3 details the process of optimising and validating the FOXO1 assay in order to reproducibly quantify the active FOXO1 levels of single cells from a range of different cell lines. The assay was validated by modulating the active FOXO1 levels using a small molecule PI3K inhibitor. This resulted in an increase in the measured active FOXO1 as expected. The specificity of the detection antibody was confirmed using western blot as an orthogonal technique. Further validation of the FOXO1 MAC chip assay was conducted using FOXO1 siRNA knockdown, which resulted in an expected reduction in the active FOXO1 signal.
Chapter 4 focuses on the application of the FOXO1 assay in measuring FOXO1 target engagement. This was conducted using the well-established cellular thermal shift assay (CETSA), using a TIRF-based readout to achieve single-cell resolution. 3 different FOXO1 inhibitors were utilised, which successfully displayed differing levels of target engagement. Validation of the single-cell CETSA method was conducted using the traditional western-blot CETSA technique.
Chapter 5 details the move from working with standard cell lines to studying clinically relevant patient samples in the context of COPD. Importantly, this chapter focuses on developing workflows that enable the successful use of small quantities of patient samples, an important consideration owing to the precious nature of these cells. A workflow was created in order to successfully collect small volumes of samples from a frozen core of bronchial epithelial cells. The latter portion of the chapter explores the move towards using less invasive sampling methods, with nasal sampling being identified as strong candidate for being able to probe the cellular senescence and inflammation associated with COPD. Protocol development was conducted using nasal samples collected from healthy volunteers, with the successful evaluation of FOXO1 from both T-cells and epithelial cells.