Next generation of human cancer diagnostics: Nanomaterial-based electrochemical sensors for clinically relevant exosomes and exosomal biomarkers analysis.

Abstract

Exosomes are 40-100 nm diameter membrane vesicles which are released from cells and circulate in body fluids such as blood, urine, and saliva. Being encapsulated by a lipid bilayer, these nanoscale vesicles carry a cargo of proteins, lipids, mRNA, microRNA (miRNA) and transfer this cargo to recipient cells. Exosomes play an important role in various biological processes such as intercellular signalling, coagulation, inflammation, cellular homeostasis and involved in many pathological conditions such as cancer progression and metastasis. Due to these unique properties, exosomes are being pursued as promising biomarker source for diagnosis and prognosis of various diseases. Despite excellent analytical performance of the conventional methods for exosome analysis, most of them require large amount of input samples, long assay time and cumbersome pre-processing steps. Therefore, the development of a simple, sensitive and inexpensive platform that can be used for rapid quantification and analysis of exosomes is of great importance to biology and medicine. This PhD project endeavours to engineer such translational approaches to address the aforementioned challenges for developing an inexpensive, rapid, sensitive and specific biosensor platform. The thesis initially investigates the biogenesis, functions, diagnostic, prognostic and therapeutic potential of exosomes an exosomal biomarkers followed by a comprehensive study of recent progress in exosome analysis techniques including conventional methods as well as electrochemistry-based approaches. We then report on a simple electrochemical platform for detection of disease specific exosomes present in cell culture media using commercially available extravidin-modified screen printed electrodes. The assay has a two-step design, where initially total exosome population was captured by a generic antibody and the disease specific exosomes were subpopulated using a cancer-specific antibody. All the steps were performed on a single extravidin-modified electrode and final quantification of disease-specific exosomes were done by differential pulse voltammetry readout. Subsequent to the development of this proof of concept sensor, we attempted to address the increasing demand for detecting low concentrations of disease-specific exosomes. Utilizing the capability of quantum dots to serve as signal amplifiers, we next developed a highly sensitive electrochemical approach enabled to detect 100 exo/μL and demonstrated the clinical applicability via detecting disease-specific exosomes in serum samples derived from patients with colon cancer of different stages. As not only exosomes themselves, but also exosomal RNA showed a great promise as cancer biomarker, we also developed a simple electrochemical approach for the detection of cancer-derived exosomal miRNAs by selectively isolating the target miRNA using magnetic beads pre-functionalized with the specific capture probes. The isolated targets were then directly adsorbed onto a gold electrode surface and quantified via differential pulse voltametric readout. In our final readout strategy, we developed a novel platform using nanoporous Au—NPFe2O3NC nanocubes, which enable an efficient and easy exosome isolation with a subsequent sensitive detection. To achieve this goal, we exploited the advantages of nanocubes such as supermagnetism, high electrocatalytic and peroxidase-like activity. The approach compromise both electrochemical (amperometric) and colorimetric (naked-eye) readout strategies and was enable first to isolate the bulk exosome population and then specifically detect choriocarcinoma-derived exosomes. All the readout platforms reported in this thesis have shown excellent analytical performance with high specificity and sensitivity. We also demonstrated the applicability of all assays in complex biological samples including cohort of patient samples. We hope that in near future our research efforts will be translated from lab settings to the point-of-care platform for exosome analysis which could be used in clinical settings for improving patient care.