EV trapping: Raman characterization of single tumor-derived extracellular vesicles

The search for cancer biomarkers of easy access and with diagnostic and prognostic value has led to a growing interest in very small particles that are released not only by healthy cells but also by cancer cells. These membrane bound particles, known as extracellular vesicles (EVs), may be present in body fluids of cancer patients, such as in the blood. The idea of detecting and distinguishing these tumor-derived extracellular vesicles (tdEVs) from other small particles in body fluids has motivated us to explore and develop technology that can distinguish single tdEVs from other particles. The aim of this thesis is to detect and characterize biological nanoparticles in blood, specifically tdEVs, at the single particle level. Hence, this thesis explores various methods that enable, in a novel way, the detection and chemical characterization of individual particles and the discrimination of tdEVs from other EVs and non-EV particles, such as lipoprotein particles, in a label-free manner. One method explored is the correlation of scanning electron microscopy (SEM) and Raman spectroscopy that enables the acquisition of high resolution SEM images and the spatial correlation with chemical information as obtained from Raman micro-spectroscopic imaging. Another method is the development of optical trapping and synchronized Rayleigh and Raman scattering (OT-sRRs) for the detection and characterization of single biological nanoparticles, such as tdEVs, directly in suspension and in a label-free manner. This thesis describes the implementation of various novel methods to study biological nanoparticles in blood, from cancer cells to tdEVs and from model nanoparticles to nanoparticles in the plasma of cancer patients. These developments open an avenue not only to exploit the potential of tdEVs as cancer biomarkers, but also to study other particles in body fluids and, with that, the general nanoparticle profile, which may be affected under pathological conditions such as cancer

Immunocapturing of extracellular vesicles on stainless steel for multi-modal individual characterization with correlative light, electron and probe microscopy

Here, we report a robust platform for multi-modal analysis of immuno-captured individual extracellular vesicles (EVs). Stainless steel substrates were surface-modified to covalently immobilize specific antibodies targeting proteins found on EVs. Using PDMS microchannels, EVs were selectively captured on the substrates. Next, individual EVs were retraced and correlatively characterized here using SEM, AFM and Raman Spectroscopy.</p

Immunocapturing of extracellular vesicles on stainless steel for multi-modal individual characterization with correlative light, electron and probe microscopy

Here, we report a robust platform for multi-modal analysis of immuno-captured individual extracellular vesicles (EVs). Stainless steel substrates were surface-modified to covalently immobilize specific antibodies targeting proteins found on EVs. Using PDMS microchannels, EVs were selectively captured on the substrates. Next, individual EVs were retraced and correlatively characterized here using SEM, AFM and Raman Spectroscopy.</p

Multi-modal analysis of tumor-derived extracellular vesicles immunocaptured from plasma

Extracellular vesicles have emerged in recent years as highly promising for understanding cell communication, drug delivery, and medical applications. Specifically, tumor-derived extracellular vesicles (tdEVs) have demonstrated excellent prognostic value in cancer diagnostics compared to imaging approaches. Despite the growing body of expertise regarding EVs, great challenges remain, notably in their handling and characterization. In complex media, other particles with similar characteristics may occlude measurements. Here, a platform is presented for the immunocapturing of tdEVs for identifying their origin followed by further multi-modal analysis by Raman spectroscopy, confocal microscopy and atomic force microscopy (AFM)

Organosilicon interaction with biological membranes

Poly(dimethyl siloxane) (PDMS) is the number-one used material to produce microfluidic devices, under the assumption it is biocompatible. Other organosilicon compounds, including PDMS, are ubiquitous in daily use products such as cosmetics, pharmaceuticals and even food. Their approval in these applications is based on the notion that the substance is not absorbed systemically. Here, using a range of analytical techniques, we demonstrate that a range of organosilicon compounds do interact with cell membranes and models thereof.</p