Suthar, Jugal; (2021) Novel Biosensing Approaches for Detection of Exosomal Proteins. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Exosomes are endocytic lipid membrane-bound bodies that have been shown to carry proteins associated with cancer and neurodegenerative disease. This has led to an exponential rise in research that looks to incorporate exosomal proteins as disease biomarkers within diagnostic assays. Furthermore, ubiquitous presence of exosomes in nearly all biological fluids creates the possibly of minimally invasive liquid biopsies for the patient. However, the heterogeneity of exosomes and complexity of biological source materials requires a consideration of optimal isolation protocols. More importantly, the development of effective exosome based assays is limited by the scarcity of translational characterization approaches that are capable of determining their molecular composition and physical properties in physiological fluids. The key objectives of this doctoral research was to establish a robust exosome isolation protocol from complex media, prior to sensing the exosomes on an immunosensor transduced by acoustic wave and electrochemical measurements. This work also looked to enhance these platforms from their current baseline performance, through the implementation of various surface structure modifications. A size exclusion chromatography approach was developed for the isolation of exosomes expressing CD63, Alix, CD81 and CD9 proteins, and allowed them to be extracted effectively from cell culture media, human serum and urine. Isolated exosomes were subsequently detected on a quartz crystal microbalance with dissipation (QCM-D) monitoring, after the optimisation of an affinity-based immunofunctionalisation approach. This technique displayed high sensitivity and specificity towards exosomal CD63 at clinically relevant concentrations in complex media. The QCM-D sensor was also used as a working electrode, as part of an electrochemical cell, to enable additional impedance spectroscopy analysis of exosome binding in tandem with the QCM-D response, collectively termed EQCM-D. The combination of these approaches offers a label-free, sensitive and real-time approach to exosome detection. The sensitivity of the EQCM-D platform was improved through surface formation of tuneable gold nanoparticle arrays from selective impregnation of block-copolymer templates, taking advantage of their segregation behaviour. This presented a versatile approach to tune sensor surfaces in order to improve ligand orientation and subsequent analyte binding. Similar advancements were made on silica detection surfaces through the formation of silica inverse opal crystals, with differing thicknesses, using a single-step co-assembly approach that combines a sol-gel matrix with poly(methyl methacrylate) (PMMA) spheres. Porous networks atop the sensors increased the internal surface area significantly, translating to a higher binding capacity of exosomes notwithstanding a higher degree of artefact entrapment. The results achieved through this work offer a potential for multi-modal analysis of exosomal proteins in diagnostics, underpinned by acoustic wave methodologies and nanostructured materials.

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