The design of a digital single-molecule detection platform, with direct application to single cell analysis

Abstract

We have designed and developed a novel form of biosensor with implications to both the understanding of the mechanistics of the cell function and as a biomedical diagnostic tool. As part of a platform of technologies orientated to single cell proteomics, this project focuses on the development of single molecule microarrays (SMM), which is intended to produce proteome snap shots of individual cells. The single cell proteomics platform includes technologies for microfluidics based live cell analysis, manipulation, lysis and analysis of the proteome of a single cell. Analysis of single cells to high and detailed resolution will provide both quantitative and qualitative information on discrete events and protein dynamics, usually overlooked through ensemble measurements of large populations of inhomogeneous cells. With the aim of developing a quantitative and qualitative tool oriented to single cell proteomics; we present an antibody microarray capable of simultaneous quantification of multiple proteins to the single molecule level. The principle enhancements of design enabling the capabilities described are based around single molecule detection of fluorescently labelled analytes bound to affinity patches of antibodies; the affinity patches are limited to the microscope field of view and incorporated into microfluidic devices, detection is performed with a single resolution through the use of total internal reflection fluorescence (TIRF) microscopy. The cell is handled and lysed in a microfluidic device to minimize dilution of the sample, so that the greater majority of analyte is bound to the sensing surface at equilibrium and so detectable at the surface through TIRF evanescent wave. This technology also bears significance for the detection of many forms of analyte, such as glycan profiling, direct mRNA quantification and the analysis of post translational modification (PTM). The SMM produces rapid snap shots of a proteome; this makes it suitable for rapidly changing and dynamic cell signalling cascades, of which PTM makes a significant contribution to cell fate. We present here the basic principles of design and the initial testing of the design and current setup. Also discussed are intensions and capabilities for the technology, as well inevitable potential pitfalls and intrinsic limitations.EThOS - Electronic Theses Online ServiceGBUnited Kingdo