Resumen de De la preconcentració a la detecció ràpida d'exosomes com a biomarcadors en diagnosi clínica
There is a growing demand for biomarkers that can help detect diseases at an early stage, as well as for follow-up of patients and therapeutic strategies. Exosomes could be the next big step to reach this goal. Exosomes are membrane encapsulated biological nanometric particles of endocytic origin, which are released by all types of cells. They carry a cargo of active molecules to proximal and distal cells of the body as a mechanism of physiological communication, to maintain natural homeostasis as well as pathological responses. All types of cells use exosomes for this purpose. Importantly, one of the most remarkable features is that they are present in all the biological fluids, such as blood, saliva, urine, among others. Their easy accessibility is one of the most compelling reasons for developing exosomes as clinical biomarkers. Another striking characteristic is their molecular cargo, which can be useful for diagnosis and prognosis of several conditions and diseases. During the biogenesis, components of the cell remain in the exosomes, including tetraspanins (CD9, CD63, CD81), membrane proteins, lipids and different RNA species (mRNA and microRNA) and DNA. This cargo provides a specific signature about their cellular origin and contains critical information about processes happening at different areas of the body. Although the exosomes are considered promising candidates as biomarkers to improve the current clinical diagnostic methods and to develop rapid tests, one of the main drawbacks is that they must be detected at low concentration in very complex samples. Accordingly, conventional procedures for exosome detection usually require relatively large sample volumes and involve preliminary purification and preconcentration steps by ultracentrifugation. Therefore, this thesis addresses one of the bottlenecks that should be considered to simplify the analytical procedure in the detection of exosomes: the study and development of novel solid-phase separation methods in order to avoid ultracentrifugation. This thesis studies the specific isolation of exosomes on particle-based magnetic enrichment, which can be easily coupled with emerging technologies for the rapid detection of exosomes. A rational study of the surface proteins in exosomes, which can be recognized by magnetic particles is presented. This study is initially performed by classical methods, including isolation by ultracentrifugation, and characterization by Transmission Electron Microscopy, Nanoparticle Tracking Analysis, confocal microscopy and surface protein screening by Flow Cytometry. A comparative study of the biomarker profiling of the cells and their derived exosomes is discussed, including the general tetraspanins CD9, CD63 and CD81, and the specific cancer-related receptors (CD24, CD44, CD54, CD326 and CD340). Based on this study, the exosomes are preconcentrated by immunomagnetic separation on antiCD81-modified magnetic particles in order to achieve further detection based on spectrophotometric readout and electrochemical biosensing. The effect of the serum matrix on the immunomagnetic separation is then carefully evaluated. Finally, the study of exosomes for the detection of breast cancer is also addressed in this doctoral thesis, by different diagnostic methods in different formats, including immunoassays and electrochemical biosensors, in order to improve the analytical performance and simplified the procedure. All the strategies presented here are able to discriminate healthy and breast cancer patients based on specific epithelial cancer-related biomarkers. From this dissertation it can be concluded that an increased amount in the serum of exosomes expressing epithelial cells molecular patterns as well as alkaline phosphatase activity is a promising biomarker for breast cancer patients.
