1. Microfluidic methods for single cell analysis in clinically relevant samples
- Author
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Katsanovskaja, Ksenia, Klug, David, and Willison, Keith
- Subjects
572 - Abstract
Single cell protein analysis has the potential to comprehensively profile cellular heterogeneity advancing the understanding of cell function, disease progression and drug development. Although it is a possible contributor to the discovery of novel therapy, the protein mapping on the individual cell basis is complex and poses challenges. For example, cancer is a heterogeneous disease which analysis using conventional bulk methods can potentially conceal a dangerous population like Circulating Tumour Cells (CTCs). Analysis of CTCs can only be reliably attained using single cell technologies. The Microfluidic Affinity Capture (MAC) chip is a tool that was developed in our group to study cellular heterogeneity by measuring protein abundance in single cells. Another problem in analysing cancer cells is the difficulty in obtaining samples in a non-invasive manner. This thesis reports on attempts to obtain, concentrate and analyse CTCs from blood using a composite MAC-based device. The ability to analyse single cells has advantages beyond the potential for analysing heterogeneity. Chronic Obstructive Pulmonary Disease (COPD) is a heterogeneous illness that is characterised by a chronic inflammation. Although the immune response in COPD requires investigation, it is complicated by a lack of biomarkers and methods to non-invasively collect samples. The ability to obtain and effectively analyse precious and scarce biomaterial from lungs, is greatly advantageous for both diagnosis and tracking of COPD. Here, we report on a workflow to achieve this using sputum from negative control volunteers and COPD+ patients. Also, we describe the establishment of a novel MAC chip assay targeting FOXO3 protein which is involved in regulation of processes like tumour suppression, inflammation and senescence. Therefore, it has a biomarker potential to monitor and study diseases like cancer and COPD. We developed the protocol to analyse Forkhead box 3 (FOXO3) protein expression in nasal cells from healthy donor samples that were retrieved with a non-invasive tool (NASAM, nasal synthetic absorptive matrix). Nasal cavity is a front line of exposure to inhaled pernicious substances and, it is speculated to reflect anomalous bioprocesses in lungs preceding respiratory disease development and progression. This study provides the first-time quantification of FOXO3 protein in single cells from nasal samples. This work shows the analytical capacity of the MAC chip to study cellular heterogeneity and quantify important biomarkers in single cells of clinically relevant material.
- Published
- 2020
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