An electro-optical bead-nanochip technology for the ultrasensitive and multi-dimensional detection of small extracellular vesicles and their markers

Extracellular Vesicles

ABSTRACT Small extracellular vesicles (sEVs), including exosomes, are enriched in multiomics information mirroring their parental cells. They have been investigated in health and disease and utilised in several applications from drug discovery to diagnostics. In disease diagnostics, sEVs can be sampled via a blood draw, enabling the convenient liquid biopsy of the tissue they originate from. However, few applications with sEVs have been translated into clinical practice. We developed a Nanoparticle EXOsome Sensing (NEXOS) technology, for the ultrasensitive and multi-dimensional detection of sEVs. NEXOS comprises two methods: a novel nanoelectronics method, E-NEXOS, and a high-throughput optical detection method, O-NEXOS. Both methods share the same steps for the immunocapture and antibody-labelling of sEVs and can be combined to derive differentiated detection parameters. As a proof of concept, we show the analytical detection and sensitivity of these methods in detecting pre-prepared cancer cell-derived CD9 + CD81 + and CD9 + HER2 + sEVs. Both sEV populations were diluted in PBS and spiked in processed plasma. We also provide a novel approach for the determination of target sEVs (TEVs), target epitopes in sEVs (TEPs), and epitopes per target sEV, as yet unseen from current and emerging technologies. Further, we demonstrate the higher sensitivity of O-NEXOS compared to the gold standard techniques, as well as demonstrating that E-NEXOS possesses commensurate sensitivity whilst only being powered by 36 nanogap-based sensors per nanochip. Finally, this manuscript lays the groundwork for a scalable electronics miniaturization of E-NEXOS nanochip with millions of nanogap-based sensors for the translation of NEXOS into standard clinical practice.

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Recent Publications

Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.

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