Abstract
The development of direct analyte probed nano-extraction (DAPNe), for the untargeted extraction and analysis of biomolecules from animal tissue sections is presented. DAPNe has previously been utilised for the metabolomic analysis of single cells or sub-cellular contents, a variety of forensic applications (in which the smallest extraction diameter of 6.2 µm was achieved), and the extraction of lipids and anti-tuberculosis drugs has been demonstrated from dosed animal tissue sections, with extraction diameters as low as 105 µm, from tuberculosis granulomas. The work presented in this thesis aimed to build on this state-of-the-art, by developing DAPNe as a sensitive, repeatable, and robust technique for the untargeted, spatially-resolved extraction of metabolites, peptides, or proteins from biological samples.
The DAPNe extraction process was optimised to reduce variability between extractions, without compromising the mass spectrometry (MS) analysis. This optimised extraction method reduced the variability in the extraction diameters of DAPNe extractions from a previously reported 19 % to 9 %.
A novel method for transferring the contents of a nano-capillary, following DAPNe extraction, to a chromatography vial was created. This new method negated the previous requirement for breaking the terminus of the nano-capillary tip. This reduced the potential risks that fragments posed to chromatographic instrumentation, while increasing the calculated transfer efficiency from the previously reported 60 % to 83.9 %.
DAPNe-MS was presented for the untargeted extraction and analysis of metabolites from tissue homogenates and tuberculosis granulomas, with a high of 38 unique compounds detected in a single 5 µL extraction. Additionally, the first demonstration of DAPNe-MS for the untargeted extraction and analysis of proteins and peptides is presented, with protein detection presented from extractions as low as 17.3 µm, corresponding to an 83.5 % reduction in the smallest reported extraction diameter from biological samples.