Novel methods for the detection of exosomes as biomarkers for noncommunicable diseases

Student thesis: Doctoral thesis

Abstract

This doctoral dissertation aims to contribute to the development of new tools for the early diagnosis and monitoring of non-communicable diseases. To achieve this goal, high throughput methodologies are available in the market, but the high cost and low accessibility difficult their implementation in primary healthcare centers in low- and middle-income countries. Thereby, the development and improvement of novel methods for low resource settings remains essential to reduce the burden of those diseases. In addition to the development of new, simpler, and less expensive instruments for diagnosis, the research of novel biomarkers in liquid biopsies is also very relevant. Among the different types of biomarkers, the study of exosomes as cell secreted vesicles, widely available in all biofluids, could provide direct information about physiological and pathological processes in the tissues. Exosomes are nano-sized extracellular vesicles which are currently under intensive study as potential diagnostic biomarkers for many health disorders. Particularly, they are emerging as a new biomarker in liquid biopsies for cancer, since it is known that they are profusely released by cancer cells containing their altered molecular fingerprint. The work thus addresses the study of rapid diagnostic tests (RDTs) for the analysis of exosomes as biomarkers of clinical interest. Specifically, it is focused on the improvement of the analytical parameters, experimental designs, and technologies of those RDTs. All the approaches were tested and optimized with exosomes derived from cell culture supernatants from MCF-7, MDA-MB-231 and SKBR3 cell lines, as a model of breast cancer cells. The production, isolation and purification of those cultured exosomes were firstly addressed. Then, the obtained cancer-derived exosomes were physically and molecularly characterized with standard analytical methods, such as nanoparticle tracking analysis, cryogenic transmission electron microscopy and flow cytometry. One of the main challenges dealing with exosomes in RDTs is to increase the sensitivity and improve the limits of detection of the assays. In this direction, the use of magnetic preconcentration and nucleic acid amplification techniques, as double-tagging RT-PCR, were explored for its use in this kind of samples. In this dissertation, a novel electrochemical magneto-actuated platform combining immunomagnetic separation and genosensing of double-tagged amplicons for the quantitative detection of exosomes, is reported. The approach is based on the amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcripts . The proposed RDT was tested with human serum XII samples, enabling to discriminate breast cancer patients and healthy individuals and enhancing the limits of detection. On the other hand, paper-based detection techniques, as Lateral and Vertical Flow assays, were explored to improve the simplification of the assays, able to deal with resource-limited settings. In this dissertation, the potentiality of Vertical Flow Assay (VFA) for the visual detection of exosomes is explored for the first time. This approach relies on the use of alkaline phosphatase as enzymatic reporter, producing a colorimetric signal on the surface of the paper membranes. The visual signals were quantified with an image analysis software, obtaining promising results for the characterization of protein markers on exosomes with a simple and cost-effective device. Finally, other non-conventional approaches were explored for the detection of exosomes, focused on the detection of their intrinsic enzymatic activity. The intrinsic enzymatic activity of aldehyde dehydrogenase (ALDH) was also studied as an overexpressed biomarker in cancer disease. A novel method based on nano-flow cytometry was developed for the detection of those enzymes directly inside the exosomes. This study represents the first step towards the development of a new RDT targeting ALDH intrinsic activity of exosomes for cancer diagnostics.
Date of Award4 Nov 2022
Original languageEnglish
SupervisorMaria Merce Marti Ripoll (Director) & Maria Isabel Pividori Gurgo (Director)

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