Multiplexed protein profiling of tumor-derived extracellular vesicles using an electrokinetic sensor

Small extracellular vesicles (sEVs) that range in diameter between 40 and 200 nm are secreted from almost every cell type of the human body into the extracellular space surrounding the parental cell of origin. These nano-sized, cell-derived carriers, function as effective vehicles of intercellular communication, traveling through body fluids (blood, urine, pleural effusion, etc.) and transporting several active biomolecules, genetic information such as RNAs and DNAs among others, and surface receptors between distant cells. This novel mechanism of intercellular communication has been nowadays acknowledged to play a significant role in the development of various diseases, such as cancer. Therefore, sEVs, often referred to as exosomes, being secreted by tumor cells at an early stage, can act as suitable biomarkers offering a promising option for non-invasive diagnostics, and therapy of different forms of cancer, including lung cancer. It is well know that in order to bring a device to clinical settings it is important to scan as many markers as possible, simultaneously, as this could lead to time and costs saving results. Moreover, the employment of the same device to scan different markers in parallel produces less chance of error. In this thesis, a simple, low cost and label-free electrokinetic sensor for multiplexed extracellular vesicles' detection and profiling of their biomarkers' expression, based upon the electrokinetic principle, investigated in commercially available silica capillaries is presented. The method of detection investigated the streaming current variation which is recorded before and after the interaction between the sEVs' surface markers and the capture probes immobilized on the functionalized microcapillary's inner surface. The detection was performed on sEVs originated from the non-small-cell lung cancer (NSCLC) H1975 cell line and from pleural effusions obtained from lung cancer patients, for a set of representative surface markers, such as CD63, CD9 and epidermal growth factor receptor (EGFR), the former two being markers for exosomes and the latter regarded as overexpressed in lung cancer. Experiments were performed on bare, non-functionalized capillaries as well as on micro-capillaries functionalized with capture probes to test the stability of the signal. Repeated measurements on the capture probes functionalized micro-capillaries were conducted in order to investigate the reproducibility of their signal. Comparisons between control measurements and functionalized micro-capillaries interacting with H1975-derived EVs in order to examine the influence of non-specific binding and attempts of signal enhancement were carried out for both two-channels and four-channels measurements resulting more problematic for the latter, stressing the need to further improve the multiplexed platform. Moreover, since the sensor had never been tested on clinical samples it was important to assess its performance and the experiments showed promising results. As a future step, the sensor could be tested and integrated into a microfluidic platform.